 |
   |
           |
|
 | |
|
Biochemistry Cell
Biology Computational
Biology Developmental
Biology Ecology Evolution Genetics Microbiology Molecular
Biology Plant
Biology Science
Education Structural
Biology |
Wymore, T., D.W. Ii, and J. Hempel (2007) Mechanistic implications of the cysteine-nicotinamide adduct in aldehyde dehydrogenase based on quantum mechanical/molecular mechanical simulations. Biochemistry 46:9495-9506 Recent computer simulations of the cysteine nucleophilic attack on propanal in human mitochondrial aldehyde dehydrogenase (ALDH2) yielded an unexpected result: the chemically reasonable formation of a dead-end cysteine-cofactor adduct when NAD+ was in the "hydride transfer" position. More recently, this adduct found independent crystallographic support in work on formyltetrahydrofolate dehydrogenase, work which further found evidence of the same adduct on re-examination of deposited electron densities of ALDH2. Although the experimental data showed that this adduct was reversible, several mechanistic questions arise from the fact that it forms at all. Here, we present results from further quantum mechanical/molecular mechanical (QM/MM) simulations toward understanding the mechanistic implications of adduct formation. These simulations revealed formation of the oxyanion thiohemiacetal intermediate only when the nicotinamide ring of NAD+ is oriented away from the active site, contrary to prior arguments. In contrast, and in seeming paradox, when NAD is oriented to receive the hydride, disassociation of the oxyanion intermediate to form the dead-end adduct is more thermodynamically favored than maintaining the oxyanion intermediate necessary for catalysis to proceed. However, this disassociation to the adduct could be avoided through proton transfer from the enzyme to the intermediate. Our results continue to indicate that the unlikely source of this proton is the Cys302 main chain amide. Hempel, J., H.B. Nicholas, S.T. Brown, and T. Wymore (2007) Unexpected encounters in simulations of the ALDH mechanism. Pp 9-13 in Enzymology and Molecular Biology of Carbonyl Metabolism 13, Weiner, H., E. Maser, R. Lindahl, and B. Plapp, Ed. Purdue University Press, Lafayette, IN Hempel, J., S. Stanley, J. Perozich, T. Wymore, and H.B., J.r. Nicholas (2006) Residue conservations in aldehyde dehydrogenase gene fusion products reemphasize functional interpretations. Pp 8-14 in Enzymology and Molecular Biology of Carbonyl Metabolism 12, Weiner, H., B. Plapp, R. Lindahl, and E. Maser, Ed. Purdue University Press, Lafayette, IN Wymore, T., J. Hempel, S.S. Cho, A.D. .J.r. Mackerell, H.B. .J.r. Nicholas, and D.W. .2.n. Deerfield (2004) Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism of nucleophile activation. Proteins Struct. Func. Bioinf. 57:758-771 Experimental structural data on the state of substrates bound to class 3 Aldehyde Dehydrogenases (ALDH3A1) is currently unknown. We have utilized molecular mechanics (MM) simulations, in conjunction with new force field parameters for aldehydes, to study the atomic details of benzaldehyde binding to ALDH3A1. Our results indicate that while the nucleophilic Cys243 must be in the neutral state to form what are commonly called near-attack conformers (NACs), these structures do not correlate with increased complexation energy calculated with the MM-Generalized Born Molecular Volume (GBMV) method. The negatively charged Cys243 (thiolate form) of ALDH3A1 also binds benzaldehyde in a stable conformation but in this complex the sulfur of Cys243 is oriented away from benzaldehyde yet yields the most favorable MM-GBMV complexation energy. The identity of the general base, Glu209 or Glu333, in ALDHs remains uncertain. The MM simulations reveal structural and possible functional roles for both Glu209 and Glu333. Structures from the MM simulations that would support either glutamate residue as the general base were further examined with Hybrid Quantum Mechanical (QM)/MM simulations. These simulations show that, with the PM3/OPLS potential, Glu209 must go through a step-wise mechanism to activate Cys243 through an intervening water molecule while Glu333 can go through a more favorable concerted mechanism for the same activation process. Proteins 2004. (c) 2004 Wiley-Liss, Inc. Hempel, J., J. Perozich, T. Wymore, and H.B. Nicholas (2003) An algorithm for identification and ranking of family-specific residues, applied to the ALDH3 family. Chem. Biol. Interact. 143:23-28 An algorithm for detecting amino acid residues characteristic of individual protein families from within aligned collections of paralogous sequences, and its application to the ALDH3 family versus the rest of the ALDH extended family is described. Residues illuminated by this analysis include a key intramolecular tether, a lysine that makes an intersubunit contact at the dimer interface, three residues in close association with the substrate-binding funnel, and a pair of residues suggested to participate in proton relay during the catalytic cycle. Wymore, T., D.W. Deerfield, M.J. Feidl, J. Hempel, and H.B. Nicholas (2003) Initial catalytic events in class 3 aldehyde dehydrogenase: MM and QM/MM simulations. Chem. Biol. Interact. 143:75-84 A novel enzyme mechanism has been predicted by computer simulations for formation of the thiohemiacetal intermediate in the rat ALDH3A1 enzyme. We used molecular mechanics simulations to study the atomic details of substrate binding and quantum mechanical/molecular mechanical methods to study the Cys-243 thiolate attack on benzaldehyde (BA) substrate. BA was found to produce more reactive conformers when aligned for formation of the tetrahedral thiohemiacetal in the R-configuration. In addition, the sulfhydryl proton was seen to be important for initial binding of the substrate. Finally, the free energy differences between forming a thiohemiacetal oxyanion intermediate versus forming a neutral thiohemiacetal intermediate where a proton is donated to the intermediate from the surroundings strongly favor the latter. Our results suggest that the proton donor is the amide proton from the Cys-243 backbone supported by interactions with Lys-235. Lee, J.Y.-Y., J. Hempel, and J.-S. Deng (2002) Anti-adenosine deaminase antibodies in lupus erythematosus. Lupus 11:168-174 Adenosine deaminase (ADA) is an enzyme involved in purine metabolism and has a major role in the development and function of lymphoid cells. Congenital deficiency of ADA results in severe immunodeficiency. Patients with congenital ADA deficiency treated with polyethylene glycol-conjugated bovine ADA develop antibodies to ADA. This leads us to investigate the role of anti-ADA antibodies in patients with systemic rheumatic diseases. Commercially available ADA was used in ELISA and immunoblots for detection of anti-ADA antibodies. Four out of 100 patients examined were positive for anti-ADA antibodies. Two of them had peripheral blood lymphopenia but the antibody levels did not appear to correlate with the lymphocyte counts. Immunoblotting revealed that the antibodies recognized a 40 kDa peptide of ADA, corresponding to ADA1, the major component of ADA. Affinity-purified antibodies were used to locate the distribution of ADA on Hep-2 cells and lymphocytes by indirect immunofluorescence. Anti-ADA antibodies gave a distinct nuclear speckled pattern on acetone-fixed cells. With viable cell immunofluorescence, anti-ADA antibodies also stained the cell surface of HEp-2 cells and lymphocytes, indicating surface expression of ADA. The anti-ADA antibodies failed to gain access into the cytoplasm or nuclei when added to the cultures of HEp-2 cells. In summary, this is the first report of detection of anti-ADA1 autoantibody which is a new type of ANA with discrete, speckled nuclear staining, but which may not be associated with lymphopenia. Hempel, J., R. Lindahl, J. Perozich, B. Wang, I. Kuo, and H. Nicholas (2001) Beyond the catalytic core of ALDH: a web of important residues begins to emerge. Chem. Biol. Interact 130:39-46 Site-directed mutagenesis was performed in class 3 aldehyde dehydrogenase (ALDH) on both strictly conserved, non-glycine residues, Glu-333 and Phe-335. Both lie in Motif 8 and are indicated to be of central catalytic importance from their positions in the tertiary structure. In addition, a highly conserved residue at the end of Motif 8, Pro-337, and Asp-247, which interacts with the main chain of Motif 8, were also mutated. All substitutions were conservative. Kinetic values clearly show that Glu-333 and Phe-335 are crucial to efficient catalysis, along with Asp-247. Pro-337 appears to have a different role, most likely relating to folding. Perozich, J., I. Kuo, R. Lindahl, and J. Hempel (2001) Coenzyme specificity in aldehyde dehydrogenase. Chem. Biol. Interact 130:115-124 Influences on coenzyme preference are explored. Lysine 137 (192 in class 1/2 ALDH) lies close to the adenine ribose, directly interacting with the adenine ribose in NAD-specific ALDHs and the 2'-phosphate of NADP in NADP-specific ALDHs. Lys-137 in class 3 ALDH interacts with the adenine ribose indirectly through an intervening water molecule. However, this residue is present in all ALDHs and, as a result, is unlikely to directly influence coenzyme specificity. Glutamate 140 (195) coordinates the 2'- and 3'-hydroxyls of the adenine ribose of NAD in the class 3 tertiary structure. Thus, it appeared that this residue would influence coenzyme specificity. Mutation to aspartate, asparagine, glutamine or threonine shifts the coenzyme specificity towards NADP, but did not completely change the specificity. Still, the mutants show the 2'-phosphate of NADP is repelled by Glu-140 (195). Although Glu-140 (195) has a major influence on coenzyme specificity, it is not the only influence since class 3 ALDHs, can use both coenzymes, and class 2 ALDHs, which are NAD-specific, have a glutamate at this position. One explanation may be that the larger space between Lys-137 (192) and the adenine ribose hydroxyls in the class 3 ALDH:NAD binary structure may provide space to accommodate the 2'-phosphate of NADP. Also, a structural shift upon binding NADP may also occur in class 3 ALDHs to help accommodate the 2'-phosphate of NADP. Wymore, T., H.B. Nicholas, and J. Hempel (2001) Molecular dynamics simulation of class 3 aldehyde dehydrogenase. Chem. Biol. Interact 130:201-207 Molecular dynamics (MD) simulation of the rat class 3 aldehyde dehydrogenase (ALDH) with nicotinamide dinucleotide (NAD) cofactors and explicit water molecules are reported. Our results demonstrate that MD simulation using the latest methodologies can maintain the crystal structure of the enzyme, as well as closely reproduce the short timescale dynamics of the enzyme. Furthermore, the examination of the distance between the nucleophilic Cys-243 and the NAD cofactor reveal important fluctuations that could be linked to ALDH catalysis. Finally, our quantum mechanical model of benzaldehyde in the active site of ALDH demonstrates that the enzyme requires only minor conformational changes to be poised for nucleophilic attack on the substrate. Hempel, J. (2001) An Orientation to Edman chemistry. Pp 102-122 in Practical Methods in Advanced Protein Chemistry, Brown, W.E., and G.C. Howard, Ed. CRC Press, Boca Raton Hempel, J., I. Kuo, J. Perozich, B.C. Wang, R. Lindahl, and H. Nicholas (2001) Aldehyde dehydrogenase: maintaining critical active site geometry at motif 8 in the class 3 enzyme. Eur. J. Biochem. 268:722-726 Alignment of all known, diverse members of the aldehyde dehydrogenase (ALDH) extended family revealed only two strictly conserved, nonglycine residues, a glutamate and a phenylalanine residue. Both occur in one of the highly conserved 'motif' segments and both occupy strategic locations in the tertiary structure at the bottom of the catalytic funnel. In class 3 ALDH, these are Glu333 and Phe335. In addition, Asp247, which is not highly conserved but is characteristic of class 3 ALDHs, hydrogen bonds the main chain between Glu333 and Phe335. These three residues were mutated conservatively. Michaelis constants determined for both NAD/propanal and NADP/benzaldehyde substrate pairs show all three residues to be crucial to effective catalysis, and suggest that the hydrogen bond to Asp247 is a key element in maintaining precise geometry of key elements at the active site. Perozich, J., I. Kuo, J.S. Boesch, B.C. Wang, R. Lindahl, and J. Hempel (2000) Shifting the NAD/NADP preference in class 3 aldehyde dehydrogenase. Eur. J. Biochem. 267:6197-6203 Among pyridine-nucleotide-dependent oxidoreductases, the class 3 family of aldehyde dehydrogenases (ALDHs) is unusual in its ability to function with either NAD or NADP. This is all the more surprising because an acidic residue, Glu140, coordinates the adenine ribose 2' hydroxyl. In many NAD-dependent dehydrogenases a similarly placed carboxylate is thought to be responsible for exclusion of NADP. The corresponding residue in most ( approximately 71%) sequences in the ALDH extended family is also Glu, and most of these are NAD-specific enzymes. Site-directed mutagenesis was performed on this residue in rat class 3 ALDH. Our results indicate that this residue contributes to tighter binding of NAD in the native enzyme, but suggest that additional factors must contribute to the ability to utilize NADP. Mutagenesis of an adjacent basic residue (Lys137) indicates that it is even more essential for binding both coenzymes, consistent with its conservation in nearly all ALDHs (> 98%). Hempel, J., J. Perozich, T. Chapman, J. Rose, J.S. Boesch, Z.J. Liu, R. Lindahl, and B.C. Wang (1999) Aldehyde dehydrogenase catalytic mechanism. A proposal. Adv. Exp. Med. Biol. 463:53-59 Perozich, J., H. Nicholas, R. Lindahl, and J. Hempel (1999) The big book of aldehyde dehydrogenase sequences. An overview of the extended family. Adv. Exp. Med. Biol. 463:1-7 Perozich, J., H.B. Nicholas, B.-C. Wang, R. Lindahl, and J. Hempel (1999) Relationships within the aldehyde dehydrogenase extended family. Protein Sci. 8:137-146 One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project. Liu, Z.J., J. Hempel, J. Sun, J. Rose, D. Hsiao, W.R. Chang, Y.J. Chung, I. Kuo, R. Lindahl, and B.C. Wang (1998) Crystal structure of a class 3 aldehyde dehydrogenase at 2.6 A resolution. Adv. Exp. Med. Biol. 414:1-7 Perozich, J., J. Hempel, and S.M. Morris (1998) Roles of conserved residues in the arginase family. Biochim. Biophys. Acta 1382:23 Arginases and related enzymes metabolize arginine or similar nitrogen-containing compounds to urea or formamide. In the present report a sequence alignment of 31 members of this family was generated. The alignment, together with the crystal structure of rat liver arginase, allowed the assignment of possible functional or structural roles to 32 conserved residues and conservative substitutions. Two of these residues were previously identified as functionally essential by analysis of inherited defects in the type I arginase gene. Nearly half of the conserved residues are either glycines or prolines located at critical bends in the protein structure. Most metal-coordinating residues, including one histidine and four aspartic acid residues, are strictly conserved. Two additional histidines involved in metal-binding and catalysis are conserved in all arginases and in almost all other family members. Two positions with invariant similarities may serve as indirect metal ligands. Evolutionary relationships within this family were also suggested. Vertebrate type I and II arginases appear to have developed independently from an early gene duplication event. A ureohydrolase sequence from Caenorhabditis elegans is more closely related to other arginases than previously appreciated, while unclassified enzymes from Methanococcus jannaschii and Methanothermus fervidus appear more similar to arginase-related enzymes. In addition, enzymes from Arabidopsis thaliana and Synechocystis, previously identified as arginases, more closely resemble arginase-related enzymes than currently known arginases. Hempel, J., Z.J. Liu, J. Perozich, J. Rose, R. Lindahl, and B.C. Wang (1997) Conserved residues in the aldehyde dehydrogenase family. Locations in the class 3 tertiary structure. Adv. Exp. Med. Biol. 414:9-13 Liu, J., Y.J. Sun, J. Rose, Y.J. Chung, C.D. Hsiao, W.R. Chang, I. Kuo, J. Perozich, R. Lindahl, J. Hempel, and B.C. Wang (1997) The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold. Nat. Struc. Biol. 4:317-326 The first structure of an aldehyde dehydrogenase (ALDH) is described at 2.6 Å resolution. Each subunit of the dimeric enzyme contains an NAD-binding domain, a catalytic domain and a bridging domain. At the interface of these domains is a 15 Å long funnel-shaped passage with a 6 x 12 Å opening leading to a putative catalytic pocket. A new mode of NAD binding, which differs substantially from the classic beta-alpha-beta binding mode associated with the 'Rossmann fold', is observed which we term the beta-alpha,beta mode. Sequence comparisons of the class 3 ALDH with other ALDHs indicate a similar polypeptide fold, novel NAD-binding mode and catalytic site for this family. A mechanism for enzymatic specificity and activity is postulated. Sun, J., J. Hempel, J. Perozich, J. Rose, and B.C. Wang (1995) Progress toward the tertiary structure of (class 3) aldehyde dehydrogenase. Adv. Exp. Med. Biol. 372:71-77 Perozich, J., A. Leksana, and J. Hempel (1995) UDP-glucose dehydrogenase. Structural characteristics. Adv. Exp. Med. Biol. 372:79-84 Duda, R.L., J. Hempel, H. Michel, J. Shabanowitz, D. Hunt, and R.W. Hendrix (1995) Structural transitions during bacteriophage HK97 head assembly. J. Mol. Biol. 247:618-635 Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.
Nicholas, H.B., B. persson, H. Jornvall, and J. Hempel (1995) Ethanol utilization regulatory protein: profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion. Protein Sci. 4:2621-2624 The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 146:145-158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same composition. This level of similarity fails to support the suggested gene fusion. Hempel, J., J. Perozich, H. Romovacek, A. Hinich, I. Kuo, and D.S. Feingold (1994) UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases. Protein Sci. 3:1074-1080 The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD(+)-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microhetergeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of approximately 26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases. Hempel, J., H. Nicholas, and R. Lindahl (1993) Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework. Protein Sci. 2:1890-1900 Sequences of 16 NAD and/or NADP-linked aldehyde oxidoreductases are aligned, including representative examples of all aldehyde dehydrogenase forms with wide substrate preferences as well as additional types with distinct specificities for certain metabolic aldehyde intermediates, particularly semialdehydes, yielding pairwise identities from 15 to 83%. Eleven of 23 invariant residues are glycine and three are proline, indicating evolutionary restraint against alteration of peptide chain-bending points. Additionally, another 66 positions show high conservation of residue type, mostly hydrophobic residues. Ten of these occur in predicted beta-strands, suggesting important interior-packing interactions. A single invariant cysteine residue is found, further supporting its catalytic role. A previously identified essential glutamic acid residue is conserved in all but methyl malonyl semialdehyde dehydrogenase, which may relate to formation by that enzyme of a CoA ester as a product rather than a free carboxylate species. Earlier, similarity to a GXGXXG segment expected in the NAD-binding site was noted from alignments with fewer sequences. The same region continues to be indicated, although now only the first glycine residue is strictly conserved and the second (usually threonine) is not present at all, suggesting greater variance in coenzyme-binding interactions. Blochberger, T.C., J.P. Vergnes, J. Hempel, and J.R. Hassell (1992) cDNA to chick lumican (corneal keratan sulfate proteoglycan) reveals homology to the small interstitial proteoglycan gene family and expression in muscle and intestine. J. Biol. Chem. 267:347-352 A 1.9-kb cDNA clone to chick lumican (keratan sulfate proteoglycan) was isolated by screening an expressing vector library made from chick corneal RNA with antiserum to chick corneal lumican. The cDNA clone contained an open reading frame coding for a 343-amino acid protein, Mr = 38,640. Structural features of the deduced sequence include: a 18-amino acid signal peptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 62% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I, where X represents any amino acid. Lumican contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The sequential arrangement of these repeats and their spacing suggest that this region arose by duplication. The deduced sequence shows five potential N-linked glycosylation sites, four of which are in the leucine-rich region. These sites are also potential keratan sulfate attachment sites. The cDNA clone to lumican hybridizes to a 2.0-kb mRNA found in tissues other than cornea, predominantly muscle and intestine. Radiolabeling and immunoprecipitation studies show that lumican core protein is also synthesized by these tissues. The primary structure of lumican is similar to fibromodulin, decorin, and biglycan, which indicates it belongs to the small interstitial proteoglycan gene family. The expression of lumican in tissues other than cornea indicates a broader role for lumican besides contributing to corneal transparency. Churchill, P., J. Hempel, H. Romovacek, W.W. Zhang, M. Brennan, and S. Churchill (1992) Primary structure of rat liver D-beta-hydroxybutyrate dehydrogenase from cDNA and protein analyses: a short-chain alcohol dehydrogenase. Biochemistry 31:3793-3799 The amino acid sequence of D-beta-hydroxybutyrate dehydrogenase (BDH), a phosphatidyl-choline-dependent enzyme, has been determined for the enzyme from rat liver by a combination of nucleotide sequencing of cDNA clones and amino acid sequencing of the purified protein. This represents the first report of the primary structure of this enzyme. The largest clone contained 1435 base pairs and encoded the entire amino acid sequence of mature BDH and the leader peptide of precursor BDH. Hybridization of poly(A+) rat liver mRNA revealed two bands with estimated sizes of 3.2 and 1.7 kb. A computer-based comparison of the amino acid sequence of BDH with other reported sequences reveals a homology with the superfamily of short-chain alcohol dehydrogenases, which are distinct from the classical zinc-dependent alcohol dehydrogenases. This protein family, initially discerned from Drosophila alcohol dehydrogenase and bacterial ribitol dehydrogenase, is now known to include at least 20 enzymes catalyzing oxidations of distinct substrates. Hempel, J., R. Eckey, D. Berie, H. Romovacek, D.P. Agarwal, and H.W. Goedde (1992) Human liver glutamic gamma-semialdehyde dehydrogenase: structural relationship to the yeast enzyme. Comp. Biochem. Physiol. B 102:791-793 The amino acid sequences of nine tryptic peptides (containing altogether 105 amino acids) from human liver glutamic gamma-semialdehyde of dehydrogenase (hitherto designated as ALDH4) were found to correspond, at 33-66% identity, to segments from the yeast 1-proline-5-carboxylate (P5C) dehydrogenase encoded by the PUT2 gene. Hempel, J., H. Nicholas, and H. Jornvall (1991) Thiol proteases and aldehyde dehydrogenases: evolution from a common thiolesterase precursor? Proteins 11:176-183 The C-terminal 222 residues of human liver aldehyde dehydrogenase can be aligned with the C-terminal 226 residues of a thiol protease from Dictyostelium discoideum to yield 47 residue identities, including matching active site cysteine residues. A multiple alignment with three more aldehyde dehydrogenases and three more thiol proteases yields three regions with clustered residue similarities. In the tertiary structure of papain, these three regions are in close proximity although widely separated in primary structure, and many conserved residues are located in the active site groove. The three-dimensional relationships, the common thiol ester mechanisms of the enzymes, the locations of exon boundaries in the dehydrogenase and protease genes, and the conservation of internal salt-bridging and disulfide-paired residues in papain, all appear compatible with the hypothesis of an ancestral relationship between thiol proteases and aldehyde dehydrogenases. Eckey, R., R. Timmann, J. Hempel, D.P. Agarwal, and H.W. Goedde (1991) Biochemical, immunological, and molecular characterization of a "high Km" aldehyde dehydrogenase. Adv. Exp. Med. Biol. 284:43-52 Lindahl, R., and J. Hempel (1991) Aldehyde dehydrogenases: what can be learned from a baker's dozen sequences? Adv. Exp. Med. Biol. 284:1-8 Hempel, J., J.P. Rose, I. Kuo, and B.C. Wang (1991) Rat class 3 aldehyde dehydrogenase: crystals and preliminary analysis. Adv. Exp. Med. Biol. 284:9-11 Popa, M.P., T.A. McKelvey, J. Hempel, and R.W. Hendrix (1991) Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits. J. Virol. 65:3227-3237 We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli. We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large. Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species). The four remaining proteins and the material that did not enter the gel are head components. One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell. All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants. We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation. Rose, J.P., J. hempel, R. Lindahl, and B.C. Wang (1990) Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase. Proteins 8:305-308 NAD-linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AIDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and beta = 110.5 degrees. Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the kappa = 180 section at psi = 90 degrees and phi = 71 degrees and 341 degrees, indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis. Diven, W.F., B. Vietmeier, J. Hempel, and J. Chambers (1990) Purification and N-terminal characterization of Chinchilla villidera alpha-1-antitrypsin. Comp. Biochem. Physiol. B 95:39-44 1. Chinchilla, Chinchilla villidera, alpha-1-antitrypsin has been purified to homogeneity and partially characterized according to mol. wt, amino acid and carbohydrate composition and N-terminal amino acid sequence (30 residues). 2. The mol. wt is between 52,000 and 55,000 as determined by PAGE or sedimentation equilibrium. 3. The best alignment between chinchilla, human and baboon alpha-1-antitrypsin amino acid sequences offsets the chinchilla sequence 6 positions vs the primate structures. 4. This alignment suggests potential importance of the sequence His-Glu-Gln-Glu-His at positions 11-15. 5. Additionally, the segment Leu-Ala-Glu-Phe-Ala, positions 25-29, is strictly conserved. 6. Shorter N-terminal sequences available for rat and rabbit alpha-1-antitrypsin appear to follow the offset alignment vs the primate structures. Agarwal, D.P., P. Cohn, H.W. Goedde, and J. Hempel (1989) Aldehyde dehydrogenase from human erythrocytes: structural relationship to the liver cytosolic isozyme. Enzyme 42:47-52 Human red cell aldehyde dehydrogenase (ALDH) resembles the liver cytosolic isozyme in numerous physicochemical properties. This study was undertaken to establish the structural relationship between the erythrocyte and liver ALDH isozymes. The purified red cell ALDH was S-(14C)-carboxymethylated, and cleaved with trypsin. The tryptic digest was fractionated using Sephadex and reversed-phase chromatography. All peptides analyzed were identified within the liver cytosolic enzyme structure. In each case the sequence obtained corresponds exactly to a segment from the human liver cytosolic ALDH. Thus, the erythrocyte enzyme, by virtue of its chemical and structural identity with the liver cytosolic enzyme, may serve as a suitable peripheral enzyme model to understand the cause and mechanism of alcohol abuse-related changes in liver cytosolic ALDH that has been found to be reduced in alcoholics. Hempel, J, K. harper, and R. Lindahl (1989) Inducible (class 3) aldehyde dehydrogenase from rat hepatocellular carcinoma and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated liver: distant relationship to the class 1 and 2 enzymes from mammalian liver cytosol/mitochondria. Biochemistry 28:1160-1167 Peptides from rat liver aldehyde dehydrogenase (AIDH) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment match the AIDH structure from HTC rat hepatoma cells (HTC-AIDH) at all positions examined, indicating induction of the same gene product by two independent routes. This 452 amino acid residue, class 3 AIDH structure differs substantially from the 500-residue AIDH structures isolated from normal liver cytosol (class 1) and mitochondria (class 2). Despite a 29.8% identity in 429 overlapping amino acids vs the human class 1 enzyme (27.7% vs class 2), neither the N- nor C-termini coincide, and gaps are introduced to optimize the alignment. Two residues placed in the active site of human liver AIDH by chemical modification, Cys-302 and Glu-268, are conserved in class 3 AIDH as Cys-243 and Glu-209. Cys-243/302 is the only cysteine residue conserved in all known AIDH structures. Gly-245 and Gly-250 of class 1/2 AIDHs, fitting the patterns of glycine residues in coenzyme binding fold of other dehydrogenases, are also conserved. Otherwise, Cys-49, Cys-162, and Glu-487, to which functional importance has also been ascribed, are not retained in the class 3 structure. Overall, a high conservation of Gly, Pro, and Trp and similar patterns of predicted secondary structure indicate general conservation of tertiary structure, as noted with other distantly related proteins. Three exon boundaries from the human liver mitochondria AIDH gene directly correspond to the N-terminus of the rat class 3 protein and to two of the gaps in the alignment. Kaiser, R., B. Holmquist, J. Hempel, B.L. Vallee, and H. Jornvall (1988) Class III human liver alcohol dehydrogenase: a novel structural type equidistantly related to the class I and class II enzymes. Biochemistry 27:1132-1140 The primary structure of class III alcohol dehydrogenase (dimeric with chi subunits) from human liver has been determined by peptide analyses. The protein chain is a clearly distinct type of subunit distantly related to those of both human class I and class II alcohol dehydrogenases (with alpha, beta, gamma, and pi subunits, respectively). Disregarding a few gaps, residue differences in the chi protein chain with respect to beta 1 and pi occur at 139 and 140 positions, respectively. Compared to class I, the 373-residue chi structure has an extra residue, Cys after position 60, and two missing ones, the first two residues relative to class I, although the N-terminus is acetylated like that for those enzymes. The chi subunit contains two more tryptophan residues than the class I subunits, accounting for the increased absorbance at 280 nm. There are also four additional acidic and two fewer basic side chains than in the class I beta structure, compatible with the markedly different electrophoretic mobility of the class III enzyme. Residue differences between class III and the other classes occur with nearly equal frequency in the coenzyme-binding and catalytic domains. The similarity in the number of exchanges relative to that of the enzymes of the other two classes supports conclusions that the three classes of alcohol dehydrogenase reflect stages in the development of separate enzymes with distinct functional roles. In spite of the many exchanges, the residues critical to basic functional properties are either completely unchanged--all zinc ligands and space-restricted Gly residues--or partly unchanged--residues at the coenzyme-binding pocket. Jones, D.E., M.D. Brennan, J. Hempel, and R. Lindahl (1988) Cloning and complete nucleotide sequence of a full-length cDNA encoding a catalytically functional tumor-associated aldehyde dehydrogenase. Proc. Natl. Acad. Sci., USA 85:1782-1786 To study the mechanism(s) controlling expression of the tumor-associated aldehyde dehydrogenase (tumor ALDH), which appears during rat hepatocarcinogenesis, cDNAs encoding this isozyme were cloned and identified with an antibody probe. Poly(A)-containing RNA from HTC rat hepatoma cells, which have been shown to possess high levels of tumor ALDH, was used as template to synthesize double-stranded cDNA. The cDNA was methylated to protect internal sites. Two different synthetic DNA linkers were added sequentially to the cDNA to insure correct orientation for expression from the lac promoter of pUC8. A library of 100,000 independent members carrying inserts greater than 1 kilobase was obtained. From this library, two apparently identical tumor ALDH clones, differing only in size, were identified with an indirect immunological probe. The larger of the cDNA clones identified, pTALDH, was chosen for further study. Interestingly, since tumor ALDH is a dimeric enzyme, pTALDH directs synthesis of a functional tumor ALDH in the bacterial cell. The cDNA sequence has been confirmed by comparison to the amino acid sequence of tumor ALDH purified from HTC cells. Hanson, M.S., J. Hempel, and C.C. Brinton (1988) Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein. J. Bacteriol. 170:3350-3358 Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature [London] 322:265-268). We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity. Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins. We have determined the amino acid composition and amino-terminal sequence of the adhesion protein. This sequence shows limited homology with the amino-terminal sequences of several E. coli pilins, including type 1. Jornvall, H., J. Hempel, H. von Bahr-Lindstrom, J.O. Hoog, and B.L. Vallee (1987) Alcohol and aldehyde dehydrogenases: structures of the human liver enzymes, functional properties and evolutionary aspects. Alcohol 1:13-23 All three types of subunit of class I human alcohol dehydrogenase have been analyzed both at the protein and cDNA levels, and the structures of alpha, beta 1, beta 2, gamma 1, and gamma 2 subunits are known. The same applies to class II pi subunits. Extensive protein data are also available for class III chi subunits. In the class I human isozymes, amino acid exchanges occur at 35 positions in total, with 21-28 replacements between any pair of the alpha/beta/gamma chains. These values, compared with those from species differences between the corresponding human and horse enzymes, suggest that isozyme developments in the class I enzyme resulted from separate gene duplications after the divergence of the human and equine evolutionary lines. All subunits exhibit some unique properties, with slightly closer similarity between the human gamma and horse enzyme subunits and somewhat greater deviations towards the human alpha subunit. Differences are large also in segments close to the active site zinc ligands and other functionally important positions. Species differences are distributed roughly equally between the two types of domain in the subunit, whereas isozyme differences are considerably more common in the catalytic than in the coenzyme-binding domain. These facts illustrate a functional divergence among the isozymes but otherwise similar changes during evolution. Polymorphic forms of beta and gamma subunits are characterized by single replacements at one and two positions, respectively, explaining known deviating properties. Class II and class III subunits are considerably more divergent. Their homology with class I isozymes exhibits only 60-65% positional identity. Hence, they reflect further steps towards the development of new enzymes, with variations well above the horse/human species levels, in contrast to the class I forms. Again, functionally important residues are affected, and patterns resembling those previously established for the divergently related polyol dehydrogenases are encountered. The two isozymes of human aldehyde dehydrogenase also exhibit considerable differences, with only 68% structural identity. The results show an early divergence into isozymes before the man/horse species radiation. Cys-302 is a functionally important residue and is located in one of the regions with conserved hydrophobic properties. Other regions with large differences in hydropathic properties may explain the absence of cross-hybridizing isozyme forms of human liver aldehyde dehydrogenase. Jornvall, H., J. Hempel, and B. Vallee (1987) Structures of human alcohol and aldehyde dehydrogenases. Enzyme 37:5-18 Human alcohol dehydrogenase is a dimeric zinc metalloenzyme for which forms of three classes, I, II and III, have been distinguished. Subunits hybridize within but not between classes. There are three types of subunit, alpha, beta, and gamma, in class I. The primary structures of all three forms have been established, as well as the overall properties and the effects of the amino acid substitutions between the various forms. Each subunit has 374 residues, of which 35 exhibit differences among the alpha, beta and gamma chains. Corresponding cDNA structures are also known, as are the genetic organization and details of the gene structures. Allelic variants occur at the beta and gamma loci. Corresponding amino acid substitutions have been characterized, and enzymatic differences between the allelic forms are explained by defined residue exchanges. The results also illustrate recent and repeated isozyme evolution, a subject where alcohol dehydrogenases exceptionally well offer detailed examples. Human aldehyde dehydrogenase occurs of two types, a mitochondrial and a cytosolic form. The enzymes are tetramers, do not contain functional metals, and have subunits which do not form inter-type hybrids. The primary structures have been determined, revealing a positional identity of 68% (in 500 residues) between the mitochondrial and cytosolic forms. The N-terminus is heterogeneous and is not blocked in the subunit of the mitochondrial enzyme, in contrast to that of the cytosolic enzyme or those of all the alcohol dehydrogenases (also cytosolic). A reactive cysteine residue at position 302 has been ascribed functional importance at or close to the active site, is conserved in the two aldehyde dehydrogenases, and is associated with the action of disulfiram on the enzyme. In Oriental populations, a mutant allelic variant of the mitochondrial protein with impaired enzyme function has also been characterized. Hempel, J., and H. Jornvall (1987) Functional topology of aldehyde dehydrogenase structures. Prog. Clin. Biol. Res. 232:1-14 Hoog, J.O., H. von BahrLindstrom, L.O. Heden, B. Holmquist, K. Larsson, J. Hempel, B.L. Vallee, and H. Jornvall (1987) Structure of the class II enzyme of human liver alcohol dehydrogenase: combined cDNA and protein sequence determination of the pi subunit. Biochemistry 26:1926-1932 The class II enzyme of human liver alcohol dehydrogenase was isolated, carboxymethylated, and cleaved with CNBr and proteolytic enzymes. Sequence analysis of peptides established structures corresponding to the pi subunit. Two segments from the C-terminal region unique to pi were selected for synthesis of oligodeoxyribonucleotide probes to screen a human liver cDNA library constructed in plasmid pT4. Sequence analysis of two identical hybridization-positive clones with cDNA inserts of about 2000 nucleotides gave the entire coding region of the pi subunit, a 61-nucleotide 5' noncoding region and a 741-nucleotide 3' noncoding region containing four possible polyadenylation sites. Translation of the coding region yields a 391-residue polypeptide, which in all regions except the C-terminal segment corresponds to the protein structure as determined directly by peptide analysis. With the class I numbering system, the exception concerns a residue exchange at position 368, the actual C-terminus which is Phe-374 by peptide data but a 12-residue extension by cDNA data, and possibly two further residue exchanges at positions 303 and 312. The size difference might indicate the existence of posttranslational modifications of the mature protein or, in combination with the residue exchanges, the existence of polymorphism at the locus for class II subunits. The pi subunit analyzed directly results in a 379-residue polypeptide and is the only class II size thus far known to occur in the mature protein. Hempel, J., J.O. Hoog, and H. Jornvall (1987) Mitochondrial aldehyde dehydrogenase. Homology of putative targeting sequence to that of carbamyl phosphate synthetase I revealed by correlation of cDNA and protein data. FEBS Lett. 222:95-98 Comparison of existing protein and cDNA data for human liver mitochondrial aldehyde dehydrogenase reveals deviations in two segments. They are shown to correspond exactly to localized frameshifts in the cDNA data, which are likely to have three reading errors. After correction of the cDNA frameshifts, a deduced amino acid sequence corresponds exactly to the data established at the protein level. In addition, extension of the shifted frame into the cDNA corresponding to the mitochondrial leader sequence allows reinterpretation of that sequence. The new leader sequence is consistent with characteristics of such segments of other mitochondrial protein pro-forms. Furthermore, the sequence displays a homology, when centered around the cleavage site, with the leader sequence of rat liver carbamyl phosphate synthetase I, suggesting a novel similarity between mitochondrial targeting sequences of two different enzymes. Hempel, J., K. Nilsson, K. Larsson, and H. Jornvall (1986) Internal chain cleavage and product heterogeneity during Edman degradation of isosteric peptide analogs lacking the alpha-carbonyl function. FEBS Lett. 194:333-337 A synthetic peptide analog, with one peptide carbonyl group replaced by a methylene bridge, was submitted to structural analysis by Edman degradation. Multiple cleavages were obtained in the first cycle, due to phenylthiocarbamylation of the internal secondary amine as well as spontaneous alkaline cyclization and subsequent recoupling with the Edman reagent. Three fragments from cleavage of the peptide analog after a single Edman cycle were purified by reverse-phase high- performance liquid chromatography. The results support previous observations in a novel combination. The reactions may also be important with native polypeptides since non-quantitative alkaline cyclization now encountered can mimic apparent N-terminal heterogeneity in agreement with earlier data, while quantitative cyclization can mimic loss of N-terminal residues. von Bahr-Lindstrom, H., J.O. Hoog, L.O. Heden, R. Kaiser, L. Fleetwood, K. Larsson, M. Lake, B. Holmquist, A. Holmgreen, and J. Hempel (1986) cDNA and protein structure for the alpha subunit of human liver alcohol dehydrogenase. Biochemistry 25:2465-2470 Two cDNA clones for human liver alcohol dehydrogenase (ADH) were identified, together covering 1450 nucleotides that contain the cDNA sequence of the ADH1 locus and include a coding region of 1122 nucleotides for the alpha subunit of the enzyme. In parallel, direct peptide analyses of the carboxymethylated protein also established most of the amino acid sequence. Nucleotide and peptide data were in complete agreement and show exchanges at 24 positions in the alpha relative to the beta subunit. One of the cDNA clones had a 139-nucleotide internal deletion at a position of possible interest in relation to mRNA processing, ancestral connections, or DNA replication. The structure of the alpha subunit is homologous to that of the beta and gamma subunits but has many exchanges, also of functionally important residues, explaining the different enzymatic properties. In total, 35 of 374 amino acid residues differ between the class I isozymes, and the substitutions add an extra SH group in the alpha subunit. Only in the beta-pleated sheet region of the coenzyme-binding domain is almost complete lack of substitutions noted, illustrating the importance of this region. In contrast, the active site region is far less conserved. However, similar exchanges of functional significance have also been found in distantly related alcohol and polyol dehydrogenases. Buhler, R., J. Hempel, W.a.r.t.b. Von, and H. Jornvall (1985) Human liver alcohol dehydrogenase: the unique properties of the "atypical" isoenzyme beta 2 beta 2-Bern can be explained by a single base mutation. Alcohol 2:47-51 Two allelic variant alcohol dehydrogenase isoenzymes, beta 2 beta 2- Bern and beta 1 beta 1, coded by the ADH2 locus, were isolated from human livers of Caucasian origin. They represent the "atypical" and "typical" phenotype, respectively. beta 2 beta 2-Bern has a higher specific activity and a lower pH-optimum, has a higher kM for NAD+, is less susceptible to inactivation by iodoacetate, and cannot be activated with chloride ions. In order to define the structural basis for these properties, we determined the amino acid sequence difference between the beta 2-Bern and the beta 1 polypeptide chains. Peptides were prepared by cleavages with trypsin and CNBr, and were purified by exclusion chromatography and reverse phase high performance liquid chromatography. The structural analysis showed that beta 2-Bern differs at only one position from beta 1: Arg-47 in beta 1 is substituted for His-47 in beta 2-Bern. This exchange, which is identical to that reported for the beta 2-Oriental chain, alters the binding of the pyrophosphate group of the coenzyme NAD(H), and also that of iodoacetate, thus explaining the observed differences between beta 2 beta 2-Bern and beta 1 beta 1. Hagler, A.T., D.J. Osguthorpe, P. Dauber-Osguthorpe, and J.C. Hempel (1985) Dynamics and conformational energetics of a peptide hormone: vasopressin. Science 227:1309-1315 A theoretical methodology for use in conjunction with experiment was applied to the neurohypophyseal hormone lysine vasopressin for elucidation of its accessible molecular conformations and associated flexibility, conformational transitions, and dynamics. Molecular dynamics and energy minimization techniques make possible a description of the conformational properties of a peptide in terms of the precise positions of atoms, their fluctuations in time, and the interatomic forces acting on them. Analysis of the dynamic trajectory of lysine vasopressin shows the ability of a flexible peptide hormone to undergo spontaneous conformational transitions. The excursions of an individual phenylalanine residue exemplify the dynamic flexibility and multiple conformational states available to small peptide hormones and their component residues, even within constraints imposed by a cyclic hexapeptide ring. Zimniak-Przybylska, Z., J. Hempel, J. Przybylska, and H. Jornvall (1985) Structural characteristics of a major seed albumin of Pisum sativum. Biosci. Rep. 5:799-805 The major pea seed albumin from Pisum sativum was carboxymethylated, cleaved with CNBr, and submitted to sequence analysis of the fragments in order to characterize the structural organization of the protein chains. Four major pools of largely homogeneous CNBr fragments were obtained, and likely N- and C-terminal fragments were identified. Structural analysis suggested the presence of single positions with microheterogeneities. It also revealed structures with long segments of distinct homology (52% structural identity), indicating the presence of different but related protein chains, or less likely, of repetitive structural elements within a chain. However, preparations appear largely homogeneous in protein class, and contain similar polypeptide chains of about 200 residues in mainly hydrophilic structures, with few methionine and cysteine/half-cystine residues. Hempel, J., B. Holmquist, L. Fleetwood, R. Kaiser, J. Barros-Soderling, R. Buhler, B.L. Vallee, and H. Jornvall (1985) Structural relationships among class I isozymes of human liver alcohol dehydrogenase. Biochemistry 24:5303-5307 The alpha subunit of human liver alcohol dehydrogenase has been submitted to structural analysis. Together with earlier work on the beta and gamma subunits, the results allow conclusions on the relationship of all known forms of the class I type of the enzyme. Two segments of the alpha subunit were determined; one was also reinvestigated in the beta and gamma subunits. The results establish 11 residue replacements among class I subunits in the segments analyzed and show that the alpha, beta, and gamma protein chains each are structurally distinct in the active site regions, where replacements affect positions influencing coenzyme binding (position 47; Gly in alpha, Arg in beta and gamma) and substrate specificity (position 48; Thr in alpha and beta, Ser in gamma). Residue 128, previously not detected in beta and gamma subunits, corresponds to a position of another isozyme difference (Arg in beta and gamma, Ser in alpha). The many amino acid replacements in alcohol dehydrogenases even at their active sites illustrate that in judgements of enzyme functions absolute importance of single residues should not be overemphasized. Available data suggest that alpha and gamma are the more dissimilar forms within the family of the three class I subunits that have resulted from two gene duplications. The class distinction of alcohol dehydrogenases previously suggested from enzymatic, electrophoretic, and immunological properties therefore also holds true in relation to their structures. von, B.a.h.r.-., R. Jeck, C. Woenckhaus, S. Sohn, J. Hempel, and H. Jornvall (1985) Characterization of the coenzyme binding site of liver aldehyde dehydrogenase: differential reactivity of coenzyme analogues. Biochemistry 24:5847-5851 The mitochondrial isozyme of horse liver aldehyde dehydrogenase was labeled with brominated [5-(3- acetylpyridinio)pentyl]diphosphoadenosine. Specific labeling of a coenzyme binding region was proven by an enzymatic activity of the isozyme with the nonbrominated coenzyme derivative, optical properties of the complex, stoichiometry of incorporation, and protection against inactivation. A cysteine residue was selectively modified by the brominated coenzyme analogue and was identified in a 35-residue tryptic peptide. This cysteine residue corresponds to Cys-302 of the cytoplasmic isozyme and has earlier been implicated in disulfiram binding, confirming a position close to the active site. In contrast, the butyl homologue of the coenzyme analogue labels another residue of the mitochondrial isozyme. Thus, in the same isozyme, two residues are selectively reactive. They are concluded to be close together in the tertiary structure and to be close enough to the coenzyme binding site to be differentially labeled by coenzyme analogues differing only by a single methylene group. Hempel, J., R. Kaiser, and H. Jornvall (1985) Mitochondrial aldehyde dehydrogenase from human liver. Primary structure, differences in relation to the cytosolic enzyme, and functional correlations. Eur. J. Biochem. 153:13-28 The 500-residue amino acid sequence of the subunit of mitochondrial human liver aldehyde dehydrogenase is reported. It is the first structure determined for this enzyme type from any species, and is based on peptides from treatments with trypsin, CNBr, staphylococcal Glu-specific protease, and hydroxylamine. The chain is not blocked (in contrast to that of the acetylated cytosolic enzyme form), but shows N- terminal processing heterogeneity over the first seven positions. Otherwise, no evidence for subunit microheterogeneities was obtained. The structure displays 68% positional identity with that of the corresponding cytosolic enzyme, and comparisons allow functional interpretations for several segments. A region with segments suggested to participate in coenzyme binding is the most highly conserved long segment of the entire structure (positions 194-274). Cys-302, identified in the cytosolic enzyme in relation to the disulfiram reaction, is also present in the mitochondrial enzyme. A new model of the active site appears possible and involves a hydrophobic cleft. Near- total lack of conservation of the N-terminal segments may reflect a role of the N-terminal region in signaling the transport of the mitochondrial protein chains. Non-conservation of interior regions may reflect the differences between the two enzyme forms in subunit interactions, explaining the lack of heterotetrameric molecules. The presence of some internal repeat structures is also noted as well as apparently general features of differences between cytosolic and mitochondrial enzymes. Hempel, J., and H. Jornvall (1985) Cleavage at acyl-proline bonds with sodium in liquid ammonia: application with nanomolar amounts of peptides and separation of products by high-performance liquid chromatography for structural analysis. Anal. Biochem. 151:225-230 Cleavage of X-Pro bonds with metallic sodium in liquid ammonia is a little-used method due to difficulties with handling of reagents, variable cleavage yields, and separation of peptides from salt byproducts. Construction of a small distillation/reaction apparatus permitted peptide incubations at the nanomole scale. Reverse-phase high- performance liquid chromatography (HPLC) of the residue after removal of NH3 allows separation of the salts and fractionation of the cleaved peptides which may be taken directly for sequence analysis. HPLC also allows rapid assessment of the degree of cleavage before structural analysis. Cleavage of some peptides proceeded in high yield while others were cleaved poorly or not at all, modifying earlier generalizations on factors influencing cleavage. Hempel, J., B.a.h.r.-. von, and H. Jornvall (1984) Aldehyde dehydrogenase from human liver. Primary structure of the cytoplasmic isoenzyme. Eur. J. Biochem. 141:21-35 Analysis of CNBr fragments and other peptides from human liver cytoplasmic aldehyde dehydrogenase enabled determination of the complete primary structure of this protein. The monomer has an acylated amino terminus and is composed of 500 amino acid residues, including 11 cysteine residues. No evidence of any microheterogeneity was obtained, supporting the concept that the enzyme is a homotetramer . The disulfiram-sensitive thiol in the protein, earlier identified through its reaction with iodoacetamide, is contributed by a cysteine residue at position 302, while the cysteine which in horse liver mitochondrial aldehyde dehydrogenase is reactive with coenzyme analogs appears to correspond to either Cys-455 or Cys-463. Analysis of glycine distribution and prediction of secondary structures to localize beta alpha beta regions typical for coenzyme-binding are not fully unambiguous, but suggest a short region around position 245 as a likely segment for this function. In this region, sequence similarities to parts of a bacterial aspartate-beta-semialdehyde dehydrogenase and a mammalian alcohol dehydrogenase were noted. Otherwise, no extensive similarities were detected in comparisons with characterized mammalian enzymes of similar activity or subunit size as aldehyde dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase and glutamate dehydrogenase, respectively). von, B.a.h.r.-., J. Hempel, and H. Jornvall (1984) The cytoplasmic isoenzyme of horse liver aldehyde dehydrogenase. Relationship to the corresponding human isoenzyme. Eur. J. Biochem. 141:37-42 The structural divergence between the cytoplasmic isoenzymes of aldehyde dehydrogenase from different species was investigated by analysis of peptides from the horse protein, and correlation of the results with the complete primary structure of the human isoenzyme. The amino acid sequences of these two proteins show a high degree of homology (91% of residues compared are identical). The differences observed are spread over the entire polypeptide chains, with only one cluster, which is close to a reactive cysteine residue and also adjacent to the most conserved region (covering 68 residues) in the primary structures of the whole enzymes. The secondary structure predicted for the human isoenzyme is mainly unaffected by the residue differences in the horse isoenzyme, although limited conformational changes might be compatible with an unexpected overrepresentation of differences involving isoleucine (12 of 43 exchanges represent a loss of Ile in the horse protein). Two cysteine residues that correlate with catalytic activity are identically positioned in the enzyme from the two species. Fairwell, T., H. Krutzsch, J. Hempel, J. Jeffery, and H. Jornvall (1984) Acetyl-blocked N-terminal structures of sorbitol and aldehyde dehydrogenases. FEBS Lett. 170:281-289 Two new dehydrogenase structures, the 354-residue polypeptide chain of sorbitol dehydrogenase (from sheep liver) and the 500-residue polypeptide chain of cytoplasmic aldehyde dehydrogenase (from human liver), have blocked N-termini. The N-terminal peptides were purified by reverse-phase high-performance liquid chromatography and submitted to mass spectrometry after derivatization. They were also analyzed by dipeptidyl carboxypeptidase digestion, utilizing gas chromatography- mass spectrometry for dipeptide identifications. Results are consistent and establish that sorbitol dehydrogenase has N-terminal acetylalanine and aldehyde dehydrogenase N-terminal acetylserine in amino acid sequences that are compatible with estimates from chemical analyses. The two N-terminal residues found are typical of acetylated proteins in general, extend the group of known acetylated dehydrogenases, and show that these intracellular proteins are frequently N-terminally acetylated. Duester, G., G.W. Hatfield, R. Buhler, J. Hempel, H. Jornvall, and M. Smith (1984) Molecular cloning and characterization of a cDNA for the beta subunit of human alcohol dehydrogenase. Proc. Natl. Acad. Sci., USA 81:4055-4059 Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related alpha, beta, and gamma polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the beta subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing clone, pADH12, which contained an 1100-base-pair cDNA insert, was subjected to DNA sequence analysis. The sequence indicated that the cDNA encoded information for the carboxyl-terminal 91 amino acids of a class I ADH and a 3' untranslated region of 593 nucleotides. Comparisons with the carboxyl terminus of the human ADH beta subunit indicated that the cDNA encoded the beta polypeptide. This probe may facilitate genetic studies of various human alcohol-related syndromes, as well as enable basic molecular studies on human ADH gene expression. Buhler, R., J. Hempel, W.a.r.t.b. von, and H. Jornvall (1984) Atypical human liver alcohol dehydrogenase: the beta 2-Bern subunit has an amino acid exchange that is identical to the one in the beta 2- Oriental chain. FEBS Lett. 173:360-366 The "atypical' human liver alcohol dehydrogenase dimer, homogeneous for beta 2-Bern chains, was isolated from human liver of Caucasian individuals. It is derived from an allelic variant at the ADH2 gene locus and exhibits a considerably higher specific activity and lower pH optimum than its "typical' counterpart (isoenzyme beta 1 beta 1) from the beta 1-chain predominant in Caucasians. Peptides were prepared by trypsin or CNBr cleavage, and were purified by exclusion chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Structural analysis of the peptides showed that beta 2-Bern differs at one position from beta 1. Thus, Arg-47 in beta 1 is substituted by His in beta 2-Bern. This exchange, compatible with a one-base mutation, explains all functional differences by altered interactions with the pyrophosphate moiety of the coenzyme. The difference is also structurally identical to that found for another atypical beta 2- subunit, the beta 2-Oriental type of major Asian occurrence, linking these two atypical forms of human alcohol dehydrogenase. Hempel, J., R. Kaiser, and H. Jornvall (1984) Human liver mitochondrial aldehyde dehydrogenase: a C-terminal segment positions and defines the structure corresponding to the one reported to differ in the Oriental enzyme variant. FEBS Lett. 173:367-373 A C-terminal segment of mitochondrial human liver aldehyde dehydrogenase was characterized. The results prove that a central part of this segment largely but not completely agrees with a structure of a tryptic peptide previously reported for the same isoenzyme. This part corresponds to a segment that contains the exchanged residue in the functionally deficient Oriental variant of mitochondrial aldehyde dehydrogenase [(1984) Proc. Natl. Acad. Sci. USA 81, 258-261]. The data suggest important functions for the C-terminal region of aldehyde dehydrogenase, clarify previously inconsistent results, and establish this structure in the typical enzyme, including the position corresponding to the mutation in the functional variant. Buhler, R., J. Hempel, R. Kaiser, W.a.r.t.b. von, B.L. Vallee, and H. Jornvall (1984) Human alcohol dehydrogenase: structural differences between the beta and gamma subunits suggest parallel duplications in isoenzyme evolution and predominant expression of separate gene descendants in livers of different mammals. Proc. Natl. Acad. Sci., USA 81:6320-6324 Human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) occurs in multiple forms, which exhibit distinct electrophoretic mobilities and enzymatic properties. The homogeneous isoenzymes beta 1 beta 1 and gamma 1 gamma 1 were isolated from livers of Caucasians with "typical" ADH phenotype by double ternary complex affinity chromatography and ion exchange chromatography. The differences between the beta 1 and gamma 1 subunits were determined by structural analysis of all tryptic peptides from the carboxymethylated proteins. The human beta 1 and gamma 1 chains differ at 21 of the 373 positions (5.6%). Ten tryptic peptides account for the differences. All residue substitutions are compatible with one-base mutations and result in largely unaltered properties, but five lead to charge differences. Sixteen substitutions are at positions corresponding to the catalytic domain of the well-known horse enzyme; five correspond to the coenzyme- binding domain. Substitutions adjacent to important regions may correlate with differences in coenzyme binding, substrate specificities, and active-site relationships. The residue replacements between the beta 1 and gamma 1 subunits of human ADH are not identical to the known substitutions between ethanol-active (E) and steroid- active (S) subunits of horse ADH. Thus, the duplication leading to human beta 1 and gamma 1 subunits is separate and different from that leading to equine E and S subunits. Both duplications are likely to have occurred after the ancestral separation of human and equine ADH. Of the 21 residues that are different between beta 1/gamma 1, 13 in gamma 1 but only 6 in beta 1 are identical to those of the horse E chain. This suggests a closer relationship between gamma 1 and E, although beta 1 in man and E in the horse are the subunits recovered in highest yield from liver ADH preparations. Consequently, in these two mammalian species, relative activities of genes for an isoenzyme family appear to be different. Hempel, J., R. Buhler, R. Kaiser, B. Holmquist, Z.a.l.e.n. de, W.a.r.t.b. von, B. Vallee, and H. Jornvall (1984) Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme. Eur. J. Biochem. 145:437-445 Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys- 46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges. Buhler, R., J. Hempel, R. Kaiser, Z.a.l.e.n. de, W.a.r.t.b. von, and H. Jornvall (1984) Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain. Eur. J. Biochem. 145:447-453 The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. Jornvall, H., J. Hempel, B.L. Vallee, W.F. Bosron, and T.K. Li (1984) Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme. Proc. Natl. Acad. Sci., USA 81:3024-3028 The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme. Hempel, J., H. Von Bahr-Lindstrom, and H. Jornvall (1983) Structural relationships among aldehyde dehydrogenases. Pharmacol Biochem Behav 18:117-121 Two functional regions of liver aldehyde dehydrogenase were characterized before; other structures of homologous parts from isoenzymes have now been determined to obtain further information on the isoenzyme relationships. In a 22-residue region from the horse cytoplasmic and mitochondrial isoenzymes, substitutions occur at 12 positions, including a continuous six-residue portion characterized by non-conservative changes. In contrast, the same structure from the cytoplasmic isoenzyme shows exchanges at only three positions when compared to its counterpart from human cytoplasm. A similar estimate of substitution frequency between species is obtained from a larger sampling at 236 positions. Thus, the isoenzyme difference between aldehyde dehydrogenases from the same species is about five-fold greater than the species difference between corresponding isoenzymes. Hence, the relationship between cytoplasmic and mitochondrial aldehyde dehydrogenases, while recognizable, is distant. This is compatible with the fact that a property such as high sensitivity to disulfiram is a characteristic of only the cytoplasmic isoenzyme. Hempel, J.D., D.M. Reed, and R. Pietruszko (1982) Human aldehyde dehydrogenase: improved purification procedure and comparison of homogeneous isoenzymes E1 and E2. Alcohol Clin Exp Res 6:417-425 An improved purification procedure of human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 is presented. This procedure employs only three chromatographic steps to produce homogeneous E1 and E2 isoenzymes at 60% overall yield. The isoenzymes have been tested for homogeneity by electrophoresis of native and denatured species, specific activity determinations following rechromatography, as well as mapping of tryptic and CNBr fragments. Total SH group analysis has also been done on each isoenzyme. The results show that both isoenzymes are homogeneous. Similarities between E1 and E2 isoenzymes are noted in the mobility of about 40% of tryptic fragments, total SH content, and the mobility of two CNBr fragments. The results also show considerable structural differences between the isoenzymes in that CNBr maps show fragments from E1 and E2 of different molecular weight and about 60% of tryptic fragments migrate to distinct locations. Only one of SH- containing tryptic fragments migrates to the same location in both isoenzymes. E1 and E2 each consist of subunits which migrate as single bands in both sodium dodecyl sulfate (SDS) and urea electrophoresis. While the mobility of E1 and E2 subunits in SDS gels is similar, it is different in urea, showing that subunits of E1 are distinct from those of E2 and that the isoenzymes do not share subunits. Structural similarity between isoenzymes must, therefore, result from sequence similarity within regions of distinct polypeptide chains composing E1 and E2 molecules. The results presented offer a simplified procedure for preparation of the homogeneous isoenzymes; they also suggest that E1 and E2 are products of distinct genes which probably diverged from a common genetic ancestor through gene duplication and compartmentation of the cell. Pietruszko, R., J.D. Hempel, and R.C. Vallari (1982) Chemical modification and site of interaction of human aldehyde dehydrogenase E1 with disulfiram and iodoacetamide. Prog. Clin. Biol. Res. 114:61-75 A single cysteine residue is selectively alkylated by iodoacetamide in cytoplasmic human liver aldehyde dehydrogenase (isoenzyme E1). The amino acid sequence of a 35-residue fragment containing this residue is determined, showing two additional cysteine residues and also three histidine residues. The alkylation is selective for Cys-30 of this fragment, with only little alkylation even at an adjacent residue, Cys- 29. The region examined is likely to be of significance in the reaction of this isoenzyme with disulfiram since disulfiram blocks the selective alkylation. Hempel, J., R. Pietruszko, P. Fietzek, and H. Jornvall (1982) Identification of a segment containing a reactive cysteine residue in human liver cytoplasmic aldehyde dehydrogenase (isoenzyme E1). Biochemistry 21:6834-6838 A single cysteine residue is selectively alkylated by iodoacetamide in cytoplasmic human liver aldehyde dehydrogenase (isoenzyme E1). The amino acid sequence of a 35-residue fragment containing this residue is determined, showing two additional cysteine residues and also three histidine residues. The alkylation is selective for Cys-30 of this fragment, with only little alkylation even at an adjacent residue, Cys- 29. The region examined is likely to be of significance in the reaction of this isoenzyme with disulfiram since disulfiram blocks the selective alkylation. Hempel, J.D., and R. Pietruszko (1981) Selective chemical modification of human liver aldehyde dehydrogenases E1 and E2 by iodoacetamide. J Biol Chem 256:10889-10896 An improved purification procedure of human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 is presented. This procedure employs only three chromatographic steps to produce homogeneous E1 and E2 isoenzymes at 60% overall yield. The isoenzymes have been tested for homogeneity by electrophoresis of native and denatured species, specific activity determinations following rechromatography, as well as mapping of tryptic and CNBr fragments. Total SH group analysis has also been done on each isoenzyme. The results show that both isoenzymes are homogeneous. Similarities between E1 and E2 isoenzymes are noted in the mobility of about 40% of tryptic fragments, total SH content, and the mobility of two CNBr fragments. The results also show considerable structural differences between the isoenzymes in that CNBr maps show fragments from E1 and E2 of different molecular weight and about 60% of tryptic fragments migrate to distinct locations. Only one of SH- containing tryptic fragments migrates to the same location in both isoenzymes. E1 and E2 each consist of subunits which migrate as single bands in both sodium dodecyl sulfate (SDS) and urea electrophoresis. While the mobility of E1 and E2 subunits in SDS gels is similar, it is different in urea, showing that subunits of E1 are distinct from those of E2 and that the isoenzymes do not share subunits. Structural similarity between isoenzymes must, therefore, result from sequence similarity within regions of distinct polypeptide chains composing E1 and E2 molecules. The results presented offer a simplified procedure for preparation of the homogeneous isoenzymes; they also suggest that E1 and E2 are products of distinct genes which probably diverged from a common genetic ancestor through gene duplication and compartmentation of the cell. Hempel, J.D., R.C. Vallari, and R. Pietruszko (1980) On the interaction of human liver aldehyde dehydrogenase E1 isoenzyme with disulfiram and iodoacetamide. Adv. Exp. Med. Biol. 132:41-49 The E1 isoenzyme of human liver aldehyde dehydrogenase (Km acetaldehyde = 30uM, pH 7.0), when incubated with disulfiram at a stoichiometry of four moles disulfiram/tetrameric E1, is immediately inhibited to within 10% of control activity. The inhibition is reversed by 0.1% (v/v) mercaptoethanol, indicating disulfide bridge formation. An indirect attempt to locate, on maps, a peptide binding disulfiram has yielded inconsistent results. Iodoacetamide inhibits E1 slowly; inhibition is facilitated in the presence of NAD, resulting in loss of ca. 90% of control activity. Incubation with 14C iodoacetamide labels a 16,000 dalton CNBr peptide, and a ca. 4,000 dalton tryptic cleavage product. These fragments can be equated with those which have been suggested by disulfiram. |
Download the |