Two-dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution

The 1H-NMR spectra of cobrotoxin, a neurotoxic protein isolated from Formosan cobra Naja naja atra, have been studied by two-dimensional NMR techniques. Of 62 amino acid residues in cobrotoxin, the complete assignments of 58 residues have been made. The resonances from several of the remaining residues have been identified but not yet specifically assigned. The secondary structure of an antiparallel triple- and double-stranded beta-sheet has also been determined by observing the NOE.     

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.     

Primary structure of the casein macropeptide of kappa casein of buffalo]

The complete amino acid sequence of Italian water buffalo (Bubalus arnee) caseinomacropeptide, the C-terminal fragment released from kappa-casein by chymosin, has been determined. It contains 64 amino acid residues including one phosphoserine and differs from its bovine (Bos taurus) B counterpart by 10 amino acid substitutions. The sequence of the last 11 amino acid residues of para-kappa-casein is also reported. In relation to the Ala148/Asp substitution which is responsible for the different electrophoretic behaviour of bovine kappa-caseins B and A, water buffalo kappa-casein is homologous to the bovine variant B. It is suggested that a variant Thr136-Ala148 might be the wild type of the Bos genus.     

The covalent structure of calf skin type III collagen. I. The amino acid sequence of the amino terminal region of the alpha 1(III) chain (position 1--222).

The amino terminal 227 amino acid residues of the alpha 1(III) chain contain four CNBr peptides: alpha 1(III)CB3A (79 residues), CB3B, CB3C (6 residues each), CB7 (37 residues) and CB6 (99 residues). Fragmentation of the CNBr peptides was carried out using trypsin, chymotrypsin and the protease from Staphylococcus aureus V8. The fragments obtained were isolated by a combination of molecular sieve and ion exchange chromatography. The sequence analysis was performed according to the automated Edman degradation procedure.     

Glutaraldehyde cross-linking alters the environment around Trp(29) of cobrotoxin and the pathway for regaining its fine structure during refolding.

Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra- and intermolecule cross-linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross-linking was accompanied by a change in the gross conformation of cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with cobrotoxin, Trp(29) of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross-linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde-modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme-linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp(29) of cobrotoxin and glutaraldehyde-modified derivatives was formed differently. These results suggest that glutaraldehyde cross-linking leads to a change in the microenvironment of cobrotoxin Trp(29) and alters the pathway of its fine structure formation during the refolding of cobrotoxin.     

The primary structure of Bacillus subtilis acidic ribonsomal protein B-19. Isolation and characterization of peptides and the complete amino acid sequence

The complete primary structure of Bacillus subtilis acidic protein B-L9, functionally equivalent to protein L7/L12 from E. coli, has been determined. B-L9 is composed of 122 residues and has the amino acid composition: Asp3, ASN3, Thr4, Ser3, Glu22, Gln1, Pro3, Gly11, Ala21, Val14, Ile9, Leu12, Phe2, Lys13, and Arg1. The molecular weight of B-L9 is 12,633. The amino acid sequence was determined by a combination of automated Edman degradation of the intact protein in a modified Beckman sequenator, and micro dansyl-Edman degradation of the peptides obtained from digestions with trypsin, thermolysin, Staphylococcus aureus protease, chymotrypsin and pepsin. A comparison of protein B-L9 from B. subtilis with E-L12 from E. coli shows a relatively high degree of homology.     

Analysis of binding sites in human C-reactive protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by site-directed mutagenesis

Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998-4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.     

Crystal structure of rabbit phosphoglucose isomerase, a glycolytic enzyme that moonlights as neuroleukin, autocrine motility factor, and differentiation mediator

The multifunctional protein phosphoglucose isomerase, also known as neuroleukin, autocrine motility factor, and differentiation and maturation mediator, has different roles inside and outside the cell. In the cytoplasm, it catalyzes the second step in glycolysis. Outside the cell, it serves as a nerve growth factor and cytokine. We have determined the three-dimensional structure of rabbit muscle phosphoglucose isomerase complexed with the competitive inhibitor D-gluconate 6-phosphate by X-ray crystallography at 2.5 A resolution. The structure shows that the enzyme is a dimer with two alpha/beta-sandwich domains in each subunit. The location of the bound D-gluconate 6-phosphate inhibitor leads to the identification of residues involved in substrate specificity (Ser209, Ser159, Thr214, Thr217, and Thr211). The results of previously published kinetic studies suggest that a lysine and a histidine are involved in the catalytic mechanism. The crystal structure suggests active site residues Lys518 and His388 might be these residues. In addition, the positions of amino acid residues that are substituted in the genetic disease nonspherocytic hemolytic anemia suggest how these substitutions can result in altered catalysis or protein stability.     

家蚕吡哆醛激酶基因定点突变及突变体功能

【目的】吡哆醛激酶（pyridoxal kinase,PLK）是维生素B₆关键代谢酶。前期研究克隆出家蚕Bombyx mori吡哆醛激酶cDNA,经序列比对发现几个重要且保守的氨基酸残基在此蛋白中被替换。为明确家蚕吡哆醛激酶分子若干特定位置上氨基酸残基在酶功能上的作用进行本研究。【方法】采用重叠延伸法对家蚕吡哆醛激酶Thr⁴⁷,Asn¹²¹, Ile⁵⁴, Arg⁸⁸和Trp²³⁰氨基酸残基进行定点突变, 构建表达载体pET-22b（+）-PLK并转入大肠杆菌Escherichia coli Rosetta中进行诱导表达,经亲和层析对重组蛋白进行纯化, 通过酶活性检测进行功能鉴定。【结果】家蚕吡哆醛激酶Thr⁴⁷,Ile⁵⁴和Arg⁸⁸氨基酸突变后酶的催化活力分别下降82%, 58%和85%;Asn¹²¹突变对酶的催化活力几乎没有影响;而Trp²³⁰突变导致酶丧失催化活性。【结论】本研究明确了选定氨基酸侧链基团在家蚕吡哆醛激酶催化功能上的意义。     

Comparison of the amino acid sequences of the variable regions of light chains derived from two homogeneous rabbit anti-pneumococcal antibodies

The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody to type III pneumococci was determined. This L chain, designated BS-5, exhibits a greater degree of homology with the basic sequence of human kappa chains of subgroup I (72%) than with subgroups II and III. L-chain BS-5 differs from another L chain (BS-1), also derived from an antibody to type III pneumococci (Jaton, 1974), by eight amino acid residues, even though the chains are identical within the N-terminal 30 residues. Six of these eight substitutions are located within the three hypervariable sections of the variable half: Asn/Ser in position 31, Glu/Ala in position 55, Asx/Thr, Thr/Gly, Thr/Gly and Val/Tyr in positions 92, 94, 96 and 97 respectively. The two anti-pneumococcal L chains BS-1 and BS-5 are much more similar to each other than to an anti-azobenzoate L chain (Appella et al., 1973), from which they differ by 30 and 29 residues respectively. Of these interchanges 13-15 are confined to the three hypervariable sections, and 11 occur within the N-terminal 27 positions. The three chains have an identical sequence from residue 98 to residue 139, except for a possible inversion of two residues in positions 130-131 of the anti-azobenzoate chain.     

The primary structure of cassowary (Casuarius casuarius) goose type lysozyme

The complete amino acid sequence of cassowary (Casuarius casuarius) goose type lysozyme was analyzed by direct protein sequencing of peptides obtained by cleavage with trypsin, V8 protease, chymotrypsin, lysyl endopeptidase, and cyanogen bromide. The N-terminal residue of the enzyme was deduced to be a pyroglutamate group by analysis with a LC/MS/MS system equipped with the oMALDI ionization source, and then confirmed by a glutamate aminopeptidase enzyme. The blocked N-terminal is the first reported in this enzyme group. The positions of disulfide bonds in this enzyme were chemically identified as Cys4-Cys60 and Cys18-Cys29. Cassowary lysozyme was proved to consist of 185 amino acid residues and had a molecular mass of 20408 Da calculated from the amino acid sequence. The amino acid sequence of cassowary lysozyme compared to that of reported G-type lysozymes had identities of 90%, 83%, and 81%, for ostrich, goose, and black swan lysozymes, respectively. The amino acid substitutions at PyroGlu1, Glu19, Gly40, Asp82, Thr102, Thr156, and Asn167 were newly detected in this enzyme group. The substituted amino acids that might contribute to substrate binding were found at subsite B (Asn122Ser, Phe123Met). The amino acid sequences that formed three alpha-helices and three beta-sheets were completely conserved. The disulfide bond locations and catalytic amino acid were also strictly conserved. The conservation of the three alpha-helices structures and the location of disulfide bonds were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group.     

The amino acid sequence of zinc-carboxypeptidase from Streptomyces griseus

The amino acid sequence of a zinc-carboxypeptidase from S. griseus (Cpase SG) was determined by automated Edman degradation and carboxypeptidase digestion of the S-carboxymethylated protein and by sequence analyses of peptides produced by cyanogen bromide cleavage and by lysyl endopeptidase digestion of the S-carboxymethylated protein. This enzyme is characterized by a uniquely broad substrate specificity which combines the specificities of mammalian Cpase A and Cpase B (J. Biochem. 86, 683-694, 1979). Cpase SG consists of 328 amino acid residues. The amino acid sequence of Cpase SG is partially similar to those of bovine Cpase A and Cpase B (sequence identity, 28-29%). In the sequence of Cpase SG, residues that are functionally important in mammalian Cpase A and Cpase B were all found at the corresponding positions. Residue 255 (according to the numbering system for bovine Cpase A), which, in the other Cpases, contributes to the difference in specificity between Cpase A (Ile-255) and Cpase B (Asp-255), was Asp. However, residue 254 was Ile, in contrast to Ser or Thr in all of the forms of Cpase A and Cpase B examined to date. The increase in hydrophobicity caused by the change at position 254 and the presence of negative charge at position 255 is probably one of the reasons for the broad substrate specificity of Cpase SG.     

Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction

In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction after modification with GA.     

Primary structure of pregnancy zone protein. Molecular cloning of a full-length PZP cDNA clone by the polymerase chain reaction

A full-length cDNA clone of the human pregnancy zone protein (PZP) was cloned from the hepatocellular carcinoma cell line Hep3B. Based on the exon sequences of the PZP gene (Devriendt et al. (1989) Gene 81, 325-334; Marynen et al., unpublished data), primer pairs were designed to amplify six overlapping fragments of the PZP cDNA. The obtained cDNA is 4609 bp long and contains an open reading frame coding for 1482 amino acids, including a signal peptide of 25 amino acid residues. Comparison with the published partial PZP amino acid sequence (Sottrup-Jensen et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7353-7357) and the PZP genomic sequences confirmed the identity as a PZP cDNA. 71% of the corresponding amino acid residues in PZP and human alpha 2-macroglobulin (alpha 2M) are identical and all cysteine residues are conserved. A typical internal thiol ester site and a bait domain were identified. A Pro/Thr polymorphism was identified at amino acid position 1180, and an A/G nucleotide polymorphism at bp 4097.     

Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.     

Site-directed mutagenesis of CCR2 identified amino acid residues in transmembrane helices 1, 2, and 7 important for MCP-1 binding and biological functions

Monocyte chemotactic protein-1 (MCP-1) binds its G-protein-coupled seven transmembrane (TM) receptor, CCR2B, and causes infiltration of monocytes/macrophages into areas of injury, infection or inflammation. To identify functionally important amino acid residues in CCR2B, we made specific mutations of nine residues selected on the basis of conservation in chemokine receptors and located TM1 (Tyr(49)), TM2 (Leu(95)), TM3 (Thr(117) and Tyr(120)), and TM7 (Ala(286), Thr(290), Glu(291), and His(297)) and in the extracellular loop 3 (Glu(278)). MCP-1 binding was drastically affected only by mutations in TM7. Reversing the charge at Glu(291) (E291K) and at His(297) (H297D) prevented MCP binding although substitution with Ala at either site had little effect, suggesting that Glu(291) and His(297) probably stabilize TM7 by their ionic interaction. E291A elicited normal Ca(2+) influx. H297A, Y49F in TM1 and L95A in TM2 that showed normal MCP-1 binding did not elicit Ca(2+) influx and elicited no adenylate cyclase inhibition at any MCP-1 concentration. MCP-1 treatment of HEK293 cells caused lamellipodia formation only when they expressed CCR2B. The mutants that showed no Ca(2+) influx and adenylate cyclase inhibition by MCP-1 treatment showed lamellipodia formation and chemotaxis. Our results show that induction of lamellipodia formation, but not Ca(2+) influx and adenylate cyclase inhibition, is necessary for chemotaxis.     

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.     

Amino acid sequence of the Anthopleura xanthogrammica heart stimulant, anthopleurin-B

Anthopleurin-B, the most potent peptide heart stimulant from the sea anemone Anthopleura xanthogrammica, was shown to exist as a single polypeptide chain consisting of 49 amino acid residues. The sequence of the peptide was shown to be: Gly-Val-Pro-Cys-Leu-Cys-Asp-Ser-Asp-Gly- Pro-Arg-Pro-Arg-Gly-Asn-Thr-Leu-Ser-Gly-Ile-Leu-Trp-Phe-Tyr-Pro-Ser- Gly-Cys-Pro-Ser-Gly-Trp-His-Asn-Cys-Lys-Ala-His-Gly-Pro-Asn-Ile-Gly- Trp-Cys-Cys-Lys-Lys. The carboxymethylcysteine derivative, tryptic and chymotryptic peptides (obtained from the derivative and separated by high performance liquid chromatography) were sequenced by manual Edman degradation. Although six carboxymethylcysteine residues were formed by reduction and alkylation of the polypeptide, no cysteine residues were detectable in the native protein, indicating that there are three cystine residues in anthopleurin-B. The amino acid sequence differs in 7 places from anthopleurin-A: at residues 3 (Pro for Ser), 12 (Arg for Ser), 13 (Pro for Val), 21 (Ile for Thr), 24 (Phe for Leu), 42 (Asn for Thr), and 49 (Lys for Gln). These differences are important since anthopleurin-B is about a 12.5-fold better heart stimulant than anthopleurin-A from A. xanthogrammica, anthopleurin-C from Anthopleura elegantissima, and toxin II from Anemonia sulcata.     

The solution structure of the cytokine-binding domain of the common beta-chain of the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5

The haemopoietic cytokines, granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5 bind to cell-surface receptors comprising ligand-specific alpha-chains and a shared beta-chain. The beta-chain is the critical signalling subunit of the receptor and its fourth domain not only plays a critical role in interactions with ligands, hence in receptor activation, but also contains residues whose mutation can lead to ligand-independent activation of the receptor. We have determined the NMR solution structure of the isolated human fourth domain of the beta-chain. The protein has a fibronectin type III fold with a well-defined hydrophobic core and is stabilised by an extensive network of pi-cation interactions involving Trp and Arg side-chains, including two Trp residues outside the highly conserved Trp-Ser-Xaa-Trp-Ser motif (where Xaa is any amino acid) that is found in many cytokine receptors. Most of the residues implicated in factor-independent mutants localise to the rigid core of the domain or the pi-cation stack. The loops between the B and C, and the F and G strands, that contain residues important for interactions with cytokines, lie adjacent at the membrane-distal end of the domain, consistent with their being involved cooperatively in binding cytokines. The elucidation of the structure of the cytokine-binding domain of the beta-chain provides insight into the cytokine-dependent and factor-independent activation of the receptor.     

Rat liver alcohol dehydrogenase of class III. Primary structure, functional consequences and relationships to other alcohol dehydrogenases

The amino acid sequence of alcohol dehydrogenase of class III from rat liver (the enzyme ADH-2) has been determined. This type of structure is quite different from those of both the class I and the class II alcohol dehydrogenases. The rat class III structure differs from the rat and human class I structures by 133-138 residues (exact value depending on species and isozyme type); and from that of human class II by 132 residues. In contrast, the rat/human species difference within the class III enzymes is only 21 residues. The protein was carboxymethylated with iodo[2(14)C]acetate, and cleaved with CNBr and proteolytic enzymes. Peptides purified by exclusion chromatography and reverse-phase high-performance liquid chromatography were analyzed by degradation with a gas-phase sequencer and with the manual 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. The protein chain has 373 residues with a blocked N terminus. No evidence was obtained for heterogeneity. The rat ADH-2 enzyme of class III contains an insertion of Cys at position 60 in relation to the class I enzymes, while the latter alcohol dehydrogenase in rat (ADH-3) has another Cys insertion (at position 111) relative to ADH-2. The structure deduced explains the characteristic differences of the class III alcohol dehydrogenase in relation to the other classes of alcohol dehydrogenase, including a high absorbance, an anodic electrophoretic mobility and special kinetic properties. The main amino acid substitutions are found in the catalytic domain and in the subunit interacting segments of the coenzyme-binding domain, the latter explaining the lack of hybrid dimers between subunits of different classes. Several substitutions provide an enlarged and more hydrophilic substrate-binding pocket, which appears compatible with a higher water content in the pocket and hence could possibly explain the higher Km for all substrates as compared with the corresponding values for the class I enzymes. Finally the class III structure supports evolutionary relationships suggesting that the three classes constitute clearly separate enzymes within the group of mammalian zinc-containing alcohol dehydrogenases.