Structural Studies of Alcohol Dehydrogenase from Human Liver

Transcription

1 Enr. J. Biochem. 25 (1972) Structural Studies of Alcohol Dehydrogenase from Human Liver Hans JORNVALL and Regina PIETRUSZKO Medicinska Nobelinstitutet, Biokemiska Avdelningen, Karolinska Institutet, Stockholm (Received October ll/november 22, 1971) Tryptjc peptide maps of human liver alcohol dehydrogenase show this protein to be homologous to the corresponding protein of the horse. A subunit molecular weight very close to is established for the human enzyme. Sequence analysis of about one quarter of the tryptic peptides of human alcohol dehydrogenase show identical residues to the horse enzyme at about goo/,, of the positions, which is considered fairly representative of the general resemblance between the two proteins. All amino acid exchanges found are compatible with one-base mutations in the genetic code. Two different types of subunits of human liver alcohol dehydrogenase are identified. They are essentially similar but differ at some positions, one of which is No. 43 (valine in one subunit and alanine in the other). Still other subunit types may exist. The known occurrence of isoenzymes of human liver alcohol dehydrogenase may therefore be explained at least in part by subunits of different primary structures. The amino acid differences between the subunits of the human enzyme are not found at the same positions as those between the two types of subunit of the horse enzyme. The structural differences between subunits from the two species seem greater than between the subunits within either species. Isoenzyme differences may, therefore, have evolved independently in the two species. The suggestion that some of the amino acid exchanges between the horse subunits may be directly involved in the substrate binding is supported. The isoenzyme and species differences at position 43 is only three residues away from the reactive active site cysteine residue. The region around this important residue is thus not kept constant in liver alcohol dehydrogenase during evolution. Alcohol dehydrogenase from human liver occurs in multiple forms. A three-group isoenzyme pattern similar to the one found in the horse liver enzyme has been observed on gel-electrophoresis [i] as well as more complex patterns [2,3]. The isoenzyme distribution varies among different livers examined and a developmental change has been reported [Z]. In addition, isoenzyme forms indistinguishable from others upon electrophoresis but detectable due to different kinetic properties are known [4,3]. The subunit composition and relationship of all these isoenzymes is unknown and it is difficult to correlate the various forms reported in different studies. Human liver alcohol dehydrogenase preparations have been purified [5-7,3]. The molecular weight is reported to be [5] and the active enzyme to be a dimer [8]. No structural studies, except for a total amino acid composition [7], have, however, been performed on human liver alcohol dehydrogenase. It is therefore not known whether isoenzymes occur due to the presence of subunits with different Unusual Abbreviation. CM- = carboxymethyl-. Enzymes. Alcohol dehydrogenase (EC ); glyceraldehyde-3-phosphate dehydrogenase (EC ) ; trypsin (EC ). primary structures, as in the horse enzyme [9], or whether other factors alone modify primarily homogeneous subunits to produce varying isoenzymes. Neither is the relationship between the human and horse enzymes known. Available molecular weight determinations [5] suggest the human enzyme to be somewhat larger than the equine form but their subunits, after monomerisation, can nevertheless be polymerised to produce hybrids [8]. In the present work the structure of human liver alcohol dehydrogenase was studied with the aim of comparing its primary structure with that of the horse enzyme. At the same time the human dehydrogenase preparation was examined for evidence of structurally different subunits that could explain the occurrence of isoenzymes. The extent of structural similarity between the human and equine enzymes has been established. Subunits with different primary structures were found in the human enzyme and their relationship towards the different subunits of the horse enzyme ascertained. In addition, the structural differences found between the human and equine enzymes help to explain the function of certain regions of the protein molecule of liver alcohol dehydrogenase.

2 284 Structural Studies of Human Liver Alcohol Dehydrogenase Eur. J. Biochem. MATERIALS AND METHODS Preparation of [14C]Carboxymethylated Enzyme Proteins Human liver alcohol dehydrogenase was prepared as described by Pietruszko and Theorell [3]. Isoenzyme fractions 1 and 2, which were present in all human livers examined [3], were pooled after the CM-cellulose chromatography and passed through a column of Sephadex G-100. This preparation contained isoenzymes 1 and 2 in about equal amounts as judged by starch-gel electrophoresis [3]. The material was freeze-dried, dissolved in 6 M guanidine-hc1 containing 2 mm EDTA and 0.1 M Tris ph 8.10 (10 mg protein/ml solution), reduced with dithioerythritol (0.5 pmol/mg protein, corresponding to about 45O/, molar excess over protein SH-groups) and carboxymethylated with iodo- [2-14C]acetate (1.58 pmol/mg protein, equivalent to about iso/, molar excess over total SH-groups) at 20 "C for 3 h. After repeated dialysis against 1 mm HC1 the carboxymethylated protein solution (3 mg/ml) was used directly for subsequent experiments. Horse liver alcohol dehydrogenase (EE isoenzyme) was kindly supplied by Dr Akeson in our laboratory. The freeze-dried protein was carboxymethylated as described above. Preparation of Peptide Maps Trypsin was obtained from the Worthington Biochemical Corporation and added to the carboxymethylated protein solution in 1 mm HCl, after which ammonium bicarbonate (final concentration lo/o, w/v) was added in solid form. The ratio of trypsin to protein was 1 : I00 by weight and digestion was performed at 37 "C for 4 h. The material was then freeze-dried. Peptide mapping was performed on Whatman No. 3 MM paper by high-voltage electrophoresis in cooled tanks [lo] and chromatography in n-butano1 -acetic acid- water-pyridine (15: 3 : 12 : 10, v/v/v/v) as previously used with alcohol dehydrogenase [9]. Dimensions were changed by stitching appropriate areas onto new papers [ll] and after each step the resolution was controlled by autoradiography. The first dimension was electrophoresis at ph 6.5; the second dimension was chromatography for basic peptides, electrophoresis at ph 3.5 for acidic peptides and electrophoresis at ph 1.9 followed by chromatography as a third dimension for neutral peptides [9,12]. Papers were finally stained with ninhydrin [13] or specific reagents for arginine, tyrosine and tryptophan [14]. Determination of Amino Acid Sequences Peptides were purified by paper electrophoresis and chromatography in the systems mentioned above. The total composition of pure peptides was obtained with a Beckman-Spinco model 120-B amino acid analyser after hydrolysis at 110 "C with 6 N HC1 containing 1 o/o phenol for 24 h. End groups were determined with the dansyl technique [15] and sequence analysis was performed with the dansyl-edman method [16,17]. Dansyl amino acids were identified by thin-layer chromatography on polyamide sheets [18] in four systems as previously described [19]. RESULTS Comparison between the Tryptic Digests of the [14C]Carboxymethylated Alcohol Dehydrogenases from Human and Horse Liver The [14C]carboxymethylated human liver alcohol dehydrogenase preparation (3 mg) as well as the corresponding derivative of horse liver alcohol dehydrogenase (EE-isoenzyme) were digested with trypsin under identical conditions. The peptide digests obtained were then mapped by electrophoresis and chromatography as described in the previous section. In this way most peptides were clearly resolved. The neutral peptides, stained with ninhydrin, are shown in Fig. 1 and the corresponding autoradiograph in Fig. 2. The peptide maps obtained from the human and the horse enzymes are very similar. In most cases a peptide spot in one enzyme can be matched with a corresponding spot in the other. The approximate number of spots and therefore tryptic peptides is the same in both enzymes. Peptides containing particular residues, like CM-cysteine, revealed by autoradiography (cf. Fig. 2), or arginine and tyrosine, revealed by specific staining, are also very similar. In addition, the N- and C-terminal peptides of the horse enzyme can be matched with identically placed peptide spots in the digest of human liver alcohol dehydrogenase. Each digest has two peptides containing tryptophan. One is basic, common to both, the other is neutral in the digest of the human enzyme but acidic in that of the horse. It is, however, interesting to notice that the neutral human peptide containing tryptophan is identical in mobility to the corresponding peptide [9] found in the other type, SS, of horse isoenzyme. All these facts establish that the structures of the enzyme from the two different species are closely related. Therefore, the subunit size of human liver alcohol dehydrogenase is similar to the one of the equine enzyme, and all human subunits are, between themselves, similar in structure. A more detailed inspection of the peptide maps reveal that a few of the spots from the digest of human liver alcohol dehydrogenase are stained considerably weaker than the surrounding peptide spots. They are also proportionately weaker than the

3 Vo1.25, No.2, 1972 H. JORNVALL and It. PIETRUSZKO 285 Fig. 1. A two-dimensional resolution of the neutral peptides from the digests o/ [14C]carboxymethylated horse (right) and submitted to electrophoresis at ph 1.9. This paper was then stitched for the final dimension, chromatography in human (left) alcohol dehydrogenase. After electrophoresis butanol-acetic acid-water-pyridine (15: 3: 12: 10, v/v/ at ph 6.5 of the original digests the paper areas containing v/v) onto a new sheet in such a way that the two fingerthe neutral peptides were stitched onto a new sheet and prints would form mirror images of each other. Peptides were revealed by staining with ninhydrin Fig.2. An autoradiograph of the peptide maps shown in Fig.1. The arrows indicate the peptide containing the carboxymethylated active-site cysteine residue of the horse enzyme and the two analogous peptides (A and B) of the human enzyme corresponding spots in the digest of the equine enzyme. In one case this difference is also clearly noticable in the strength of the spots on the autoradiographs (cf. Fig.2). This applies to the peptide spot containing cysteine No. 46 of the horse enzyme [19], which is indicated by an arrow in Fig.2. That spot is divided into two spots, located closely together (A and B in Fig. 2) in the map of the human enzyme. Moreover, each of these two spots have approximately half the density of the surrounding spots.

4 286 Structural Studies of Human Liver Alcohol Dehydrogenase Enr. J. Biochem. Such a picture is exactly what one expects to find when a protein preparation contains two types of subunits which are identical except for differences at just a few positions. In this case the general spot density corresponds to peptides which are common to all subunits whereas split and correspondingly weaker spots are dirived from the few peptides that differ among the subunits. It may also be recalled that this situation was indeed found when digests of the hybrid isoenzymes EX of horse liver alcohol dehydrogenase were studied [B]. Peptide spots peculiar to the E- or S-subunits are then recovered in half the yield of the common spots (cf. Fig.1 in [B]). The differences in the case of the horse subunits do not, however, affect the same tryptic peptides as in the case of the human enzyme. These results suggest that human liver alcohol dehydrogenase may contain more than one type of subunit, which might explain the occurrence of isoenzymes ; that the different subunits are essentially similar except at a few positions, one of which is close to cysteine No. 46; and that the differences between the human subunits are mainly other than those that occur between the E- and S-subunits of the horse enzyme. Amino Acid Sequence Analysis of Peptides from Human Liver Alcohol Dehydrogenase In order to verify the conclusions drawn above some of the peptides from human liver alcohol dehydrogenase were prepared pure. 15mg of the tryptic digest of the [14C]carboxymethylated protein was then submitted to paper electrophoresis on Whatman No. 3 MM paper at ph 6.5. Autoradiographs and appropriate guide-strips were used to follow the purification through the subsequent steps of electrophoresis at ph 3.5 or 1.9 and chromatography in n-butanol-acetic acid-water-pyridine (15 : 3 : 12 : 10, v/v/v/v). The purified tryptic peptides were selected to represent those of special interest. They were the two neutral peptides (called N and C) which occupy the same positions in the peptide map of the human enzyme as the N- and C-terminal tryptic peptides in the map of the horse enzyme, the two neutral peptides A and B in Fig.2 corresponding to the peptide containing the active site cysteine residue [20,21] at position No. 46 [19] in the horse enzyme, three neutral peptides (TNl, TN2 and TN3) containing CM-cysteine, two basic peptides (TB1 and TB2) and the most acidic major tryptic peptide (TA1). These peptides from the human enzyme represent some with exactly the same position in the fingerprints as the corresponding peptides of the horse enzyme as well as others whose positions do not match. The purified peptides comprise nearly onefourth of the total number of major tryptic peptides and about one-sixth of the subunit length. These peptides should therefore give a reasonable indication of the structure of the human enzyme. The number of purification steps, the recovery, the electrophoretic mobility at ph 6.5, the total composition and the N-terminus of each peptide is shown in Table 1. Peptide N. No free N-terminal amino acid of peptide N could be detected by the dansyl method and the charge at ph 6.5 is neutral although the peptide contains one basic and no acidic amino acid residues (Table 1). An N-terminal blocking group is therefore probable. The peptide could not be degraded by the Edman method anti sequence analysis was performed by partial acid hydrolysis in 9.3 N HC1 for 24 h at room temperature. Several fragments were produced, and separated by electrophoresis. The structures of these, as judged by end group analysis and by paper electrophoresis at ph 1.9 after total hydrolysis, were: Ser-(Thr,Ala), Thr-(Ala,Gly,Lys), Gly-Lys and Lys. In addition, the de-blocked free peptide Ser-(Thr,Ala,Gly,Lys) was obtained. The amino acid sequence X-Ser-Thr- Ala-Gly-Lys is thus deduced. The N-terminal blocking group is likely to be an acetyl group, as in the horse enzyme [19], but the material was not enough for an acetyl group determination. The results prove that peptide N is the N-terminal tryptic peptide of human liver alcohol dehydrogenase. Peptide C. This contains five residues. The complete amino acid sequence was determined by the dansyl-edman method to be Thr-Val-Leu-Thr-Phe in agreement with the total composition and charge (cf. Table 1). This tryptic peptide contains no basic residues and is therefore considered to be derived from the C-terminus of the protein chain of human liver alcohol dehydrogenase. This is strongly supported by the clear homology with the corresponding peptide of the horse enzyme (cf. below). Peptides A and B (cf. Fig.2). Both peptides contain eight residues (Table 1). Degradation by the dansyl-edman method showed the order of the six first residues to be Met-Val-Ala-Val-Gly-Ilein peptide A and Met-Val-Ala-Ala-Gly-Ile- in peptide B. This is in agreement with the total compositions (Tablel), which also show both peptides to contain CM-cysteine and arginine in addition. Arginine is C-terminal as both peptides are obtained from a tryptic digest. The primary structure of A is therefore Met-Val-Ala-Val-Gly-Ile-Cys(Cm)-Arg and of B Met-Val-Ala-Ala-Gly-Ile-Cys(Cm)-Arg. The two peptides differ in only the fourth position (valine in A, alanine in B). This has no effect on the electrophoretic mobilities but the increased hydrophobicity of valine as compared to alanine makes peptide A run slightly farther than B in the chromatographic system (cf. Fig.2). The two peptides are clearly homologous and their structures confirm the conclu-

5 Vo1.25, N0.2,1972 H. JORNVALL and R. PIETRUSZKO 287 Table 1. Data for ten tryptic peptides purified from hhumn liver alcohol dehydrogenase Hydrolyses were performed in 6 N HCI (with lo/, phenol) at 110 "C for 24 h. The values given are molar ratios (those below 0.3 have been omitted). No corrections for destruction, incomplete hydrolysis or impurities have been made Peptide N C A B TN1 TN2 TN3 TB1 TB2 TA1 Recovery (O/,) No. ofpurificationsteps Electrophoretic mobility at ph 6.5 [23] Composition : CM-Cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Arginine _ - _ 1.3 (1) 2.0 (2) _ (1) 2.2 (2) 1.2 (1) _ (2) 1.3 (1) - _ 0.5 (1)a 0.5 (1)s - _ 1.0 (1) 1.0 (1) 4.0 (4) 1.0 (1) (2) 2.2 (2) 2.3 (2) 1.2 (1) 2.9 (3) 1.0 (1) (1) Total N-terminus None Thr Met Met Val Cys(Cm) Cys(Cm) Ala Lys Glu a Sequence analysis reveals that the peptide contains Val-Ile. Incomplete hydrolysis of this bond explains the low recovery of Val and Ile in TS1. sion that peptides A and B originate from identical positions in two different subunits. Structural evidence is thus obtained that all protein chains of human liver alcohol dehydrogenase are not identical; at least two types with different but similar primary structures are synthesized. The valine/alanine exchange is compatible with a onebase mutation in the genetic code [22]. Peptides TN1, TN2 and TN3. These peptides contain 11, 2 and 2 residues, respectively. All were degraded by the dansyl-edman method to show the complete amino acid sequences, Val-Ile-Pro- Leu-Phe-Thr-Pro-Gln-Cys(Cm)-Gly-Lys for TN1, Cys(Cm)-Arg for TN2 and Cys(Cm)-Lys for TN3, in full agreement with the total compositions (Table 1). The amide group in TN1 is evident from the electrophoretic mobility [23] at ph 6.5 (Table 1). Peptides TB1, TB2 and TAl. Peptide TB1 contains six and peptide TB2 eight residues (Table 1). Both were degraded by the dansyl-edman method to show the complete amino acid sequences, Ala-Ala-Gly-Ala-Ala-Arg for TB1 and Lys-Pro-Ile- Gln-Glu-Val-Leu-Lys for TB2, in agreement with the total compositions (Table 1). The amide group in TB2 was positioned by determinations of the electrophoretic mobility 1231 at ph 6.5 of samples 19 Eur. J. Biochem., Vo1.25 withdrawn before and after the Edman steps removing the dicarboxylic residues. Peptide TAl contains 14 residues (Table 1) and the order of the first 12 was determined by the dansyl-edman method. The amino acid sequence obtained was in agreement with the total composition, which also showed the remaining two residues to be tyrosine and lysine. Lysine must be C-terminal as it as a tryptic peptide and the amino acid sequence obtained is then Glu- Leu- Gly- Ala-Thr-Glx -Cys( Cm)-Ile- Asx-Pro- Glx- Asx- Tyr-Lys. The electrophoretic mobility (Table 1) shows that the peptide contains two amide groups [23]. The mobility of a sample withdrawn after the first Edman step showed the first residue to have a free carboxyl group but sufficient material was not available for positioning the two amide groups on the remaining dicarboxylic acid residues. Comparison between the Structures of Alcohol Dehydrogeme from Human and Horse Liver The homology between liver alcohol dehydrogenases from these two species, already inferred from the peptide maps, can now be more accurately outlined. In Table 2 the structures of the ten peptides N,

6 288 Structural Studies of Human Liver Alcohol Dehydrogenase Eur. J. Biochem. Table 2. Comparison between the structures of ten tryptic peptides of human liver alcohol dehydrogenase and the corresponding regions of the horse enzyme Residues constituting species differences are printed in bold type. Numbers above the residues refer to the positions in the protein chain of horse liver alcohol dehydrogenase [24]. The alternative residues at positions 94 and 101 indicate the known differences [9] between the E-subunit (above) and the S-subunit (below) of the horse enzyme Horse:..-Cys(Cm)-Lys-..-Met-Val-Ala-Thr-Gly-Ile-Cys(Cm)-Arg-. Human : Acetyl-Ser-Thr-Ala-Gly-Lys-..-Cys(Cm)-Lys-..-Met-Val-Ala-Val -Gly-Ile-Cys(Cm)-Arg-... c N + +-TN A- +..-Met-Va.1-Ala-Ala-Gly-Ile-Cys(Cm)-Arg-... c -B Val-Ile-Pro-Leu-Phe-Thr-Pro-Gln-Cys(Cm)-Gly-Lys-Cys(Cm)-Arg-...-Ala-Ala-Gly-Bla- Ala- Arg-... Ile...-Val-Ile-Pro-Leu-Phe-Thr-Pro-Gln-Cys(Cm)-Gly-Lys-Cys(Cm)-Arg-...-Ala- Ala-Gly- Ala-Ala-Arg-... c TN1 + +TN2-+ C- TB Glu- Val -Gly-Ala-Thr-Glu-Cys(Cm)-Val-Asn-Pro-Gln-Asp-Tyr-Lys-Lys-Pro-Ile-Gln-Glu-Val-Leu-Thr-....-Glu-Leu-Gly-Ala-Th-Glx-Cys(Cm)- Ile -Asx-Pro-Glx-Asx-Tyr-Lys-Lys-Pro-Ile-Gln-Glu-Val-Leu-Lys-... c TA1 ++- TB Thr-Ile-Leu-Thr-Phe....-Thr-Val-Leu-Thr-Phe. C-C- -+ Ser.. C, A, B, TN1, TN2, TN3, TB1, TB2 and TAl, reported above, are compared with the corresponding regions of the horse enzyme. The results show that the protein chains of human liver alcohol dehydrogenase are, within narrow limits, identical in size to those of the horse enzyme; the terminal peptides, the peptide containing the active site cysteine residue and seven other peptides analysed are either identical or strictly homologous in the enzymes from the two different species. No deletions or insertions have been detected. The subunit size of human liver alcohol dehydrogenase is thus about, [24] and the dimeric molecular weight which is lower than the previously reported value [5]. The peptides contain 61 residues and amino acid differences are found at only 5 positions. The degree of similarity between the two enzymes in the regions investigated is thus 92 Ole. Most amino acid exchanges are highly conservative and all are compatible with one-base mutations in the genetic code [22]. The threonine/lysine exchange at position 255 (Table 2) explains one of the marked differences in the tryptic peptide maps. Due to the lysine residue at this position the basic peptide TB2 is peculiar to the peptide map of the human enzyme whereas this region in the horse enzme is covered by a larger, almost insoluble peptide [24]. The other amino acid differences detected only affect neutral residues and the three valine/isoleucine/leucine exchanges at positions 235, 241 and 371 are not detectable in the peptide maps. Subunit Differences in the Human Enzyme Peptides A and B establish the presence of two types of subunits in human liver alcohol dehydrogenase. They differ by a valinelalanine exchange at the position corresponding to No. 43 in the protein chain of the horse enzyme. The two alternative sequences occur in approximately equal amounts as judged both by the peptide maps (Fig.2) and the recoveries (Table 1). Split and weak spots in the fingerprint experiments also indicate that differences occur at a few other positions. The strengths of these split spots are not always half the general spot density as in the case of peptides A and B. The presence of more types of subunits in different relative amounts is therefore not excluded. The subunit differences found in human liver alcohol dehydrogenase are not identical to those detected in the horse enzyme. DISCUSSION Species Variation The purified peptides from human liver alcohol dehydrogenase represent nearly one quarter of the tryptic peptides and include both those (e.g. N and TN1) which are indistinguishable from the corre-

7 Vol.25, No.2,1972 H. JORNYALL and R. PIETRUSZKO 289 sponding horse peptides in the peptide maps and those (e.g. A and TR2) which differ in the fingerprint experiments. It is therefore probable that the identity of 92 between these regions of the enzymes from the two different species should be a fairly good estimation of the total relatedness between their structures. This figure gives an indication of the rate of evolutionary divergence in liver alcohol dehydrogenase. The high degree of similarity between the human and equine enzymes is also supported by the fact that all differences found are compatible with one-base mutations. Isoenzynies The presence of subunits with different primary structures offers an explanation for the occurrence of isoenzymes of human liver alcohol dehydrogenase. Two different types of subunit were found, and additional types are not excluded, in the enzyme preparation investigated. The two subunits identified are essentially similar but differ at some positions. One of these is where valine and alanine are found in about equimolar ratio at the position corresponding to No. 43 in the sequence of the horse protein chain. The enzyme preparation studied contained isoenzymes 1 and 2 [3] in about equal amounts and this may be related to the two types of protein chain detected. Neither of these isoenzymes is, however, homogeneous [3] although they have no common subunits [3]. A full investigation of the subunit differences and isoenzyme relationships will need a further separation of all isoenzymes which is not obtained with the present purification method. The valinelalanine difference between the human subunits is not present in the two types of horse subunits which contain threonine at this position (No. 43 [19]). Also, two of the amino acid exchanges (at positions 94 and 101 [9]) between the E and S subunits of the horse enzyme correspond to the human peptides TN1 and TN2, respectively (Table 2), which show no evidence of heterogeneity and are recovered in good yield (Table I). It may thus be concluded that the subunit differences of the human enzyme on the one hand and the horse E and S subunits on the other are found at different positions. In addition, the structural differences between subunits from the two species seem greater than those between the subunits within either species, although peptide maps do not reveal all differences between similar structures. Isoenzymes may therefore have evolved independently in the two species. Even in the case of the human enzyme, however, different chromosomal alcohol dehydrogenase loci seem likely, as in the horse [9] ; developmental changes are known [2] and all adult livers examined [25,2,3] have had a complicated 10 isoenzyme picture with at least two forms, isoenzyme 1 and 2 [3], which does not support a pure allelic variation. Structure-Punction Relatiomhips The isoenzymes of human liver alcohol dehydrogenase do not possess the substrate specificity difference [3] against ethanol and steroid alcohols which the horse isoenzymes have. The latter variation, therefore, seems attributable to the particular amino acid differences between the horse E and S subunits, which support the earlier conclusion [9] that at least some of these amino acids may be directly involved in the substrate binding. Finally, it may be noticed that the structural difference between the human and horse enzymes at position 43 (Table 2) is only three residues away from the reactive active site cysteine residue (No. 46). Other differences close to this position are also found in the rat enzyme [26]. This variability around the reactive cysteine residue in alcohol dehydrogenase of mammal species is in contrast to the constant region around the active site cysteine residue in another dehydrogenase, glyceraldehyde- 3-phosphate dehydrogenase, of widely different species [27,28]. Although the active site cysteine residues are selectively reactive towards iodoacetate in both enzymes and essential to the activity [21], the evolutionary differences in the surrounding regions may indicate that the reactive cysteine residues in these two dehydrogenases have different functions. The authors are indebted to Professor H. Theorell for much valuable help and support. 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8 290 H. JORNVALL and R. PIETRUSZKO: Structural Studies of Human Liver Alcohol Dehydrogenase Eur. J. Biochem. 13. Heilmann, J., Barrollier, J., and Watzke, E., Hoppe- Seyler s 2. Physiol. Chem. 309 (1957) Ambler, R. P., Biochem. J. 89 (1963) Gray, W. R., and Hartley, B. S., Biochem. J. 89 (1963) 379 and 59P. 16. Gray, W. R., Methods Enzymol. 11 (1967) Gray, W. R., and Smith, J. F., Anal. Biochem. 33 (1970) Woods, K. R., and Wang, K.-T., Biochim. Biophys. Acta, 133 (1967) Jornvall, H., Eur. J. Biochem. 14 (1970) Li, T.-K., and Vallee, B. L., Biochemistry, 3 (1964) Harris, I., Nature (London), 203 (1964) Nirenberg, M., Leder, P., Bernfield, M., Brimacombe, R., Trupin, J., Rottman, P., and O Neal, C., Proc. Nut. A d. Sci. U. S. A. 53 (1965) Offord, R. E., Nature (London), 211 (1966) Jornvall, H., Eur. J. Biochem. 16 (1970) von Wartburg, J. P., and Schiirch, P. M., Ann. N.Y. Acad. Sci. 151 (1968) Jornvall, H., and MarkoviE, O., unpublished results. 27. Allison, W. S., Ann. N.Y. A d. Sci. 151 (1968) Perham, R. N., Biochem. J. 111 (1969) 17. H. Jornvall Kemiska Institutionen I, Karolinska Institutet Solnavagen 1, S Stockholm 60, Sweden R. Pietrusxko s present address: Center of Alcohol Studies, Rutgers University New Brunswick, New- Jersey 08903, U.S.A.