©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression and Kinetic Characterization of Recombinant Human Stomach Alcohol Dehydrogenase
ACTIVE-SITE AMINO ACID SEQUENCE EXPLAINS SUBSTRATE SPECIFICITY COMPARED WITH LIVER ISOZYMES (*)

(Received for publication, October 5, 1994; and in revised form, December 7, 1994)

Natalia Y. Kedishvili (§) William F. Bosron Carol L. Stone Thomas D. Hurley Cara F. Peggs Holly R. Thomasson Kirill M. Popov Lucinda G. Carr Howard J. Edenberg Ting-Kai Li

From the Departments of Biochemistry and Molecular Biology and of Medicine, Indianapolis, Indiana 46202-5122

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (-ADH) was isolated from a human stomach cDNA library. The 373-amino acid -ADH encoded by this cDNA was expressed in Escherichia coli. The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25 °C, calculated from active-site titration of NADH binding, was 92 ± 9 units/mg. Kinetic analysis of the catalytic efficiency (k/K) of recombinant -ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human -ADH exhibited high catalytic efficiency for oxidation of all-trans-retinol to all-trans-retinal. This pathway is important in the synthesis of the transcriptional regulator all-trans-retinoic acid. Secondary alcohols and 3beta-hydroxysteroids were inactive with -ADH or were oxidized with very low efficiency. The K of -ADH for ethanol was 25 mM, and the K for primary straight chain alcohols decreased substantially as chain length increased. There are important amino acid differences in the alcohol-binding site between the human class IV () and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high k for ethanol, and the low catalytic efficiency for secondary alcohols of -ADH relative to beta(1)-ADH. For example, modeling the binding of all-trans-retinol in the human beta(1)-ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site. The deletion of Gly-117 in human -ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.


INTRODUCTION

Humans produce as many as nine different subunits in the family of dimeric NAD- and zinc-dependent alcohol dehydrogenases (ADH(^1); EC 1.1.1.1). All of these enzymes exhibit broad substrate specificity. These polypeptides are encoded by six different genes, two of which are polymorphic. The relationships between active-site structure and substrate/inhibitor specificity of the active dimers formed from eight of these subunits (alpha, beta(1), beta(2), beta(3), (1), (2), , and ) have been well characterized(1, 2) . Those containing alpha-, beta-, -, and -subunits have relatively high catalytic efficiency for ethanol oxidation and even higher catalytic efficiency for oxidation of long chain primary alcohols. The isozymes have been grouped into class I (alpha, beta, and ), class II (), and class III (), depending, in part, upon substrate specificity(1, 2) .

Recently, a new isozyme called -ADH (or µ-ADH) was isolated from human stomach tissue(3, 4, 5) . -ADH exhibits a high K for ethanol (29 mM) and a remarkably high k (1500 min) for ethanol oxidation(4, 5) . This enzyme may be responsible, in part, for ``first pass'' alcohol metabolism prior to distribution of ethanol into the systemic circulation(6) . Isozymes with similar electrophoretic mobilities are found in the human esophagus(7) , rat (8) and mouse (9) stomach, and rat retina(10) . -ADH has been classified as class IV. The amino acid sequence of the rat stomach enzyme (8) and a partial amino acid sequence of the human stomach enzyme (5) have been published. A complete coding nucleotide sequence for the human stomach enzyme assembled from partial cDNA clones(11, 12) , a partial genomic clone(11) , and fragments of the cDNA sequences amplified by PCR from a cDNA library (12, 13) were reported recently. Analysis of the deduced amino acid sequence of human stomach ADH indicates that it has 59-69% sequence identity to the other four human ADH classes (11) and that class IV ADHs likely diverged after the avian/amphibian split (8) .

In this paper, we describe the isolation of the complete cDNA encoding human stomach -ADH, the expression of an active recombinant enzyme in a heterologous system, and the characterization of the catalytic properties and substrate specificity of recombinant human -ADH. The cDNA is positively identified by the expression of an active recombinant isozyme and by the perfect match of the deduced amino acid sequence with that of -ADH peptides. Comparison of the amino acids in the substrate-binding site of -ADH and the human liver beta(1)-isozyme suggests explanations for the high catalytic efficiency of -ADH with all-trans-retinol.


EXPERIMENTAL PROCEDURES

A complete cDNA encoding human class IV ADH was isolated from a ``5`-stretch'' human stomach cDNA library (CLONTECH)(14) . The coding region of -ADH was amplified by PCR with the primers CTTTTTCGGATCCATGGGCACTGCTGGAAAAG (sense) and CCACTTGAATTCTCAAAACGTCAGGACCGT (antisense). The primers contained recognition sequences (underlined) for the restriction endonucleases BamHI and EcoRI, respectively. After cleavage, the amplification product was subcloned into the expression vector pGEX-2T (Pharmacia Biotech Inc.). The final construct of the -ADH cDNA fused with the glutathione S-transferase cDNA contained codons for 3 extra amino acids (Gly, Ser, and the starting Met) on the N terminus of -ADH. The fusion protein (glutathione S-transferase and -ADH) was expressed and purified using glutathione-agarose affinity chromatography(14, 15) . After cleavage of the fusion product with thrombin, -ADH was separated from glutathione transferase by elution from a Mono Q column (Pharmacia Biotech Inc.) with a linear salt gradient. Fractions containing active -ADH were combined and concentrated. Purity of the enzyme was evaluated by SDS-PAGE and staining for protein with Coomassie Blue(14) . Total protein concentration was determined by a dye binding assay (Bio-Rad) using bovine serum albumin as a standard(16) .

Kinetic constants for alcohols, except retinol, were determined by monitoring the production of NADH at 340 nm ( = 6.22 mM cm). The kinetic constants for all-trans-retinol were obtained by monitoring the production of all-trans-retinal at 400 nm ( = 29.5 mM cm)(17, 18) . Reaction mixtures contained 2.4 mM NAD in 0.1 M sodium phosphate, pH 7.4, at 25 °C. The concentration of active sites was determined by observing fluorescence (excitation wavelength at 328 nm) at 425 nm while titrating enzyme (1-2 mg/ml) with NADH in the presence of 99 mM isobutyramide in 10 mM sodium phosphate at pH 7.4. The concentration of -ADH active sites was evaluated from the intersection point of the linear regression of the fluorescence titration above and below NADH saturation(19) . The V(max) and K values for alcohol substrates (at 2.4 mM NAD) were calculated from a fit of the kinetic data to the Michaelis-Menten equation (V = V(max)A/(K + A), where A is the concentration of the varied substrate). The k (min) was obtained by dividing V(max) by the concentration of active sites assuming a subunit M(r) of 40,000. The K values for NAD (A) and ethanol (B) were extrapolated to saturation of the second substrate by a fit of the kinetic data to a sequential Bi Bi mechanism (V = V(max)AB/(K(A)B + K(B)A + K(A)K(B) + AB))(20) . The inhibition constant for 4-methylpyrazole was determined in 0.1 M sodium phosphate buffer containing 2.4 mM NAD, pH 7.4, at 25 °C by varying both ethanol and 4-methylpyrazole concentrations. The K of 4-methylpyrazole was calculated from a fit of the kinetic data to the equation for competitive inhibition (V = V(max)B/(K(B) (1 + I/K) + B), where B and I are ethanol and 4-methylpyrazole concentrations, respectively)(20) . Aldehyde-reducing activity was measured in 0.1 M sodium phosphate buffer, pH 7.4, at 25 °C employing 0.2 mM NADH with acetaldehyde (0.25-60 mM) and 2 mM NADH with 4-trans-(N,N-dimethylamino)cinnamaldehyde (0.5-60 mM). Acetaldehyde reduction was measured by the change of NADH absorbance at 340 nm ( = 6.22 mM cm). The reduction of 4-trans-(N,N-dimethylamino) cinnamaldehyde was followed at 398 nm ( = 42 mM cm).

The protein sequences of -ADH, rat class IV ADH, and beta(1)-ADH were aligned by the progressive alignment method of Feng and Doolittle(21) . The alignment uses the algorithm of Needleman and Wunsch (22) and the minimum mutation matrix of Dayhoff et al.(23) for scoring. Gaps are introduced by comparing the most closely related pair of sequences and are retained by the ``once a gap always a gap rule.''

The substitutions occurring in human -ADH were model-built into the human beta(1)-structure using the molecular graphics program QUANTA (Molecular Simulations, Inc.). The deletion at position 117 was modeled manually by altering local main chain dihedrals to reconnect the main chain atoms. All residues in this loop remain in allowed regions of a Ramachandran plot. Following substitution of all amino acid side chains in the dimer, the model structure was subjected to 100 cycles of energy minimization using X-PLOR 3.1 with the x-ray energy term omitted(24) . Docking of the retinol molecule into the human beta(1)-structure was performed using the program AUTODOCK (25) .


RESULTS

To clone the human stomach -ADH cDNA, a PCR amplification strategy employing inosine residues in primers was devised to reduce the degeneracy. After two rounds of amplification using nested sets of primers, an 873-bp PCR product was obtained and found to encode human stomach -ADH(14) . A human stomach cDNA library was screened with this PCR product, and a 1966-bp cDNA for human stomach -ADH was isolated (Fig. 1). The cDNA encodes the complete -ADH subunit of 373 amino acids with a M(r) of 39,902. The 5`-end of this cDNA starts with the ATG codon immediately followed by the coding region. The 3`-untranslated region is 825 bp long and contains a single polyadenylation signal at nucleotide 1877.


Figure 1: Nucleotide sequence and deduced peptide sequence of -ADH. Amino acid residues that were previously determined in our laboratory by peptide sequencing (5) are in boldface. Peptide sequences that were used for the design of degenerate oligonucleotide primers are underlined. The polyadenylation signal AATAAA is underlined. The termination codon TGA is indicated (***). The starting Met is present at position 19 of the 1966-bp clone. The 18 bp of the 5`-noncoding sequence found in the clone (not shown) are apparently a cloning artifact because they do not appear in the 5`-genomic sequence reported by Satre et al.(11) . The cDNA sequence is numbered from the initiating ATG codon as position 19. The amino acid sequence is numbered from the Gly following the initiating Met codon in accord with numbering of all other human ADH isozymes.



The -ADH cDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Approximately 1-3 mg of purified -ADH was obtained per liter of bacterial culture. The fusion protein (glutathione S-transferase and -ADH) was purified by affinity chromatography on a glutathione-agarose column. After thrombin cleavage, -ADH was purified by ion-exchange chromatography on a Mono Q column. The recombinant enzyme was homogeneous, with an apparent subunit M(r) of 40,000 on SDS-PAGE(14) . The mobility of recombinant -ADH was slightly more cathodic than that of native human -ADH on starch gel electrophoresis(14) .

To characterize the functional properties of the recombinant enzyme, the steady-state kinetics for alcohol oxidation and aldehyde reduction were examined. The specific activity of purified recombinant -ADH based on a dye-binding protein assay (16) was 47 ± 10 units/mg for three separate preparations. However, calculation of specific activity by active-site titration that directly measures the concentration of NADH-binding sites (19) resulted in a specific activity of 92 ± 9 units/mg. The kinetic constants of recombinant -ADH for oxidation of a variety of alcohols are shown in Table 1. One important property of human -ADH was the high k for ethanol oxidation (1840 min at pH 7.4). The catalytic efficiency (k/K) of -ADH for primary alcohols measured with 2.4 mM NAD increased >2 orders of magnitude as the chain length of the alcohols increased from two to six carbons in length, but the k of human -ADH was relatively constant for primary alcohols having two to six carbons (Table 1). The K values ranged from 28 mM for ethanol to 0.13 mM for hexanol. The k/K of recombinant -ADH for all-trans-retinol was 2600 min mM. The rates of oxidation of cyclohexanol and the steroid alcohols etiocholan-3beta-ol-17-one and epiandrosterone, at a maximum solubility of 200-250 mM, were <2% of that of ethanol. The apparent k/K of -ADH for acetaldehyde with 0.2 mM NADH was 2030 ± 160 min mM. -ADH also exhibited high catalytic efficiency (2900 ± 300 min mM with 2 mM NADH) toward the chromophoric aldehyde 4-trans-(N,N-dimethylamino)cinnamaldehyde, which has been used for analysis of human ADHs by stopped-flow kinetics (26) . The K of -ADH for NAD (0.21 ± 0.01 mM) was calculated from a fit of the kinetic data to the sequential Bi Bi mechanism and extrapolated to saturating ethanol concentration. The K for NAD was calculated from the same fit of the data to be 0.76 ± 0.10 mM. The K of -ADH for NADH with 150 mM acetaldehyde was 219 ± 6 µM. The K of 4-methylpyrazole calculated from a fit of the data for competitive inhibition with ethanol as the varied substrate at pH 10 was 11 ± 1 µM, and the K at pH 7.4 was 350 ± 20 µM.



Computerized docking simulations were performed with all-trans-retinol using the program AUTODOCK (25) and the x-ray structure of beta(1)-ADH (27) or the model of -ADH built by substituting the appropriate residues in the beta(1)-structure (Fig. 2). The simulations show that retinol can bind in a productive conformation in both the beta(1)-ADH (Fig. 3A) and -ADH (Fig. 3B) models. In each model, the alcohol oxygen is directly coordinated to the active-site zinc (within 2.6 Å), and the hydrogen on C-1 is in a proper orientation for hydride transfer to C-4 of the NAD nicotinamide group (3.3 Å). The first four dihedral angles of the all-trans-retinol system in the -ADH model are in the conjugated planar conformations usually observed for the alcohol (Fig. 3). However, for all-trans-retinol to bind productively to beta(1), rotations about angles 3-5 must be made as shown in Fig. 3C.


Figure 2: Alignment of -ADH with human class I beta(1)-ADH and rat stomach class IV ADH (R). The deletion of amino acid 117 in human and rat stomach ADHs is indicated (*). Amino acid numbers correspond to human class I beta(1)-ADH. Key amino acid differences mentioned under ``Results'' and ``Discussion'' are in boldface.




Figure 3: Results of retinol docking simulations using the program AUTODOCK(25) . A, all-trans-retinol docked into the substrate-binding pocket of human beta(1)-ADH. The interaction between the docked retinol molecule and the catalytic zinc atom is illustrated with a dashedline (distance of 2.2 Å). The dashedline between the betaionone ring methyl group and the carbonyl oxygen of residue 117 represents the closest contact (3.2 Å) between the docked conformation of retinol and the beta(1)-ADH substrate-binding pocket. B, all-trans-retinol docked into the model-built substrate-binding pocket of human -ADH. The dashedline illustrates the interaction between the hydroxyl oxygen of retinol and the catalytic zinc atom (distance of 2.5 Å). C, dihedral angles of all-trans-retinol docked into the beta(1)-ADH and -ADH active sites.




DISCUSSION

Our initial attempts to amplify a fragment of human stomach -ADH by PCR with degenerate oligonucleotide primers, designed according to the amino acid sequence of the purified enzyme(5) , proved unsuccessful. This suggested that the amount of -ADH mRNA in whole stomach is low. After the degeneracy of primers was reduced by incorporation of inosine residues(14) , an 873-bp PCR fragment was obtained and used to clone a 1966-bp cDNA of -ADH from a human stomach cDNA library. The deduced amino acid sequence of the cDNA shown in Fig. 1is identical to the partial amino acid sequence of -ADH (5) . The cDNA coding sequence is also identical to the cDNA and genomic sequences of -ADH published recently(11, 12, 13) .

The kinetic and molecular characterization of -ADH has been hampered by the limited quantities of suitable human stomach or esophageal tissue available for enzyme purification(4, 5) . Therefore, we cloned the -ADH coding sequence into the pGEX-2T expression vector and expressed the enzyme as a fusion protein with glutathione S-transferase in E. coli. After purification of the fusion protein by affinity chromatography, cleavage with thrombin, and further purification of -ADH by ion-exchange chromatography, a homogeneous protein preparation was obtained. The expressed protein contained 3 N-terminal amino acids (Gly, Ser, and the starting Met) that remained from the fusion protein after thrombin cleavage. While the subunit size of the expressed protein on SDS-PAGE was the same as that of the native enzyme (40 kDa), the mobility of recombinant -ADH on starch gel electrophoresis was more cathodic than that of the native enzyme. This difference in mobility could be explained by the presence of a free amino group in the recombinant enzyme and a blocked N terminus in the native enzyme. The human beta-subunit (class I) and other ADH isozyme subunits start with N-acetylated residues (28) . The specific activity of purified recombinant -ADH based on an active-site titration that directly measures the concentration of NADH-binding sites (19) was about twice that measured by a dye-binding method that estimates total protein concentration. Hence, we conclude that about half of the purified recombinant enzyme is catalytically active.

Among human isozymes, the amino acid sequence of human -ADH is most similar to that of human class I ADH(11, 12) . Fig. 2shows the comparison of -ADH and beta(1)-ADH (29) sequences by the progressive alignment method(21) . The three-dimensional structures for several enzyme-substrate complexes have been published for human beta(1)beta(1)- and beta(2)beta(2)-isozymes(27, 30) . All zinc ligands, key coenzyme-binding residues (e.g. Arg-47 and Arg-369), components of the proton relay system (e.g. Thr-48 and His-51), and important residues in the alcohol-binding site (e.g. Thr-48, Phe-93, Phe-140, and Met-306) are conserved in -ADH (Fig. 2). In spite of the similarities in overall structure of the class I and IV human ADHs, there are remarkable differences in the catalytic properties of -ADH (Table 1) compared with the class I enzymes. One important property of human -ADH is the high k for ethanol oxidation (1840 min at pH 7.4). This value is 68 times that of alphaalpha, 200 times that of beta(1)beta(1), and 21 times that of (1)(1)(31, 32) . As suggested by Farrés et al.(12) , the substitution of Asn-363 in human -ADH for His-363 in beta(1)-ADH may affect coenzyme affinity and k(27) ; coenzyme dissociation is rate-limiting for beta(1)-ADH(26) .

Purified -ADH is similar to other mammalian ADHs in that it exhibits the highest catalytic efficiency with long chain alcohols, as shown in Table 1. The increase in catalytic efficiency of -ADH with increasing chain length of primary alcohols is predominantly due to the decrease in K because the k of human -ADH remained relatively constant for primary alcohols with two to six carbons (1000-2100 min in Table 1). The decrease in primary alcohol K with increasing chain length may be due to an increase in the number of possible van der Waals interactions between the simple aliphatic primary alcohols and hydrophobic amino acids lining the deep substrate-binding site.

The kinetic properties of -ADH with more complex alcohols such as all-trans-retinol, steroid alcohols, and secondary alcohols are different from those of the class I ADHs. Vitamin A (all-trans-retinol) is oxidized to retinal and retinoic acid by alcohol and aldehyde dehydrogenases in a variety of tissues. The activity of these dehydrogenases is important for the synthesis of all-trans-retinoic acid, which regulates gene expression through binding to retinoic acid receptors(33) . It has been proposed that competitive inhibition of ADH-catalyzed retinol oxidation by ethanol could depress retinoic acid synthesis and alter gene expression, thereby causing the developmental abnormalities seen in fetal alcohol syndrome(34) . We found that -ADH purified from human stomach exhibited the highest k/K for all-trans-retinol compared with eight other human ADH isozymes(18) . The k/K of recombinant -ADH for all-trans-retinol was 2600 min mM (Table 1). The most likely sequence difference between -ADH and class I ADHs (beta(1)-ADH in Fig. 2) that could account for this difference in retinol oxidation activity is the deletion of an amino acid in a loop comprising residues 114-120 that is located on the surface of the catalytic domain. This loop clearly influences access to the alcohol-binding site as shown in the x-ray structure of human beta(1)- and beta(2)-ADHs (Fig. 3A)(29) . The length and sequence of this loop are quite variable among the enzyme classes and different species.

A docking simulation between all-trans-retinol and either human beta(1)- or -ADH explains why -ADH exhibits higher catalytic efficiency for retinol oxidation than the class I ADHs. The productive docking solution where the retinol oxygen is coordinated to the active-site zinc and C-1 of retinol is in a proper orientation for hydride transfer to the nicotinamide of NAD is shown for the beta(1)-ADH structure in Fig. 3A and for the -ADH model in Fig. 3B. The substitution of the smaller Leu in -ADH for Tyr at position 110 of beta(1)-ADH enables the change in conformation necessary to model the loop from residues 114 to 120 in -ADH shown in Fig. 3B. The conformation of bound retinol in the two enzymes is the same for the first two dihedral angles of the conjugated side chain of retinol as shown in Fig. 3C. To avoid creating unfavorable van der Waals contacts with amino acid 117 of the loop in the substrate-binding pocket of the beta(1)-structure, the remaining three dihedral angles of retinol must be rotated out of plane, thus breaking the conjugated double bond system of this chromophore. The energetic penalty to create this unfavorable retinol conformation may account for the lower k/K of the class I beta(1)-isozyme compared with that of class IV -ADH (Table 1)(18) .

A distinctive feature of human -ADH is that it does not catalyze oxidation of the 3beta-hydroxysteroids etiocholan-3beta-ol-17-one and epiandrosterone. There are two regions of the substrate-binding site (amino acid 48 and the loop at positions 114-120) that have been shown to affect 3beta-hydroxysteroid binding. Both the horse E- and S-isozymes have Ser at position 48. A deletion in the flexible loop at position 115 (DeltaAsp-115) in the S-isozyme increases the catalytic efficiency for 3beta-hydroxysteroids as compared with the E-isozyme(35, 36) . For human class I isozymes that do not have a deletion in the loop at positions 114-120, only the -isozyme with Ser-48 oxidizes 3beta-hydroxysteroids. The alphaalpha-, beta(1)beta(1)-, and -isozymes with Thr at position 48 are inactive with 3beta-hydroxysteroids(37) . Thus, it appears that the deletion of Gly-117 in the loop of -ADH is not sufficient to yield 3beta-hydroxysteroid-oxidizing activity.

One of the most surprising properties of -ADH is its inability to oxidize secondary alcohols efficiently. No activity was detected with cyclohexanol. S(+)-2-Butanol was oxidized at a measurable rate, but it had an extremely high K(120 mM) and a 2200-fold lower k/K than that of the primary alcohol homolog 1-butanol. It has been suggested that the specificity of ADHs toward secondary alcohols is affected by amino acids at positions 48 and 93(38) . However, human -ADH has the same amino acids, Thr-48 and Phe-93, as beta(1)-ADH; therefore, other amino acid substitutions must be responsible for the inactivity of -ADH with secondary alcohols. The deletion of Gly-117 in -ADH, which opens up access to the alcohol-binding site, may contribute to the inactivity of -ADH with secondary alcohols. For example, Hurley and Vessell (39) recently demonstrated that the substitution of Leu-116 in beta(1)-ADH with Ala substantially decreases the catalytic efficiency for secondary alcohols. They suggested that enlarging the entrance to the alcohol-binding site may yield fewer productive versus unproductive encounters between enzyme and secondary alcohols.

Human -ADH has high (87%) sequence identity to the rat stomach enzyme. Parés et al.(8) and Farrés et al.(12) have discussed amino acid substitutions in human class IV ADH relative to the rat class I isozymes that could account for its high k for ethanol oxidation and altered substrate specificity. Some of the exchanges in the rat stomach enzyme relative to rat class I ADH are not seen in human -ADH. For example, the Val-294 that is important in both coenzyme and substrate binding in beta(1)-ADH remains Val in human -ADH, but is Ala in rat stomach ADH (Fig. 2). The Arg-47 in beta(1)-ADH that participates in coenzyme binding remains Arg in human -ADH, but is Gly in rat stomach ADH (Fig. 2). Farrés et al.(12) concluded that the substitution of Arg-47 in the human enzyme by Gly in the rat stomach enzyme may result in the higher K for ethanol and the higher Kfor 4-methylpyrazole. The unusually high k and K of human -ADH for NAD may be due to the His-271 in human -ADH versus Arg in the class I isozymes (Fig. 2).

These kinetic studies demonstrate that the -ADH isozyme exhibits unique alcohol-oxidizing specificity relative to other classes of human ADHs. When the amino acid sequence of -ADH is compared with those of the class I isozymes, the deletion of Gly-117 and the exchange of the smaller Leu-110 in -ADH for Tyr in beta(1)-ADH may contribute to differences in substrate specificity. The docking simulations with - and beta(1)-ADHs clearly indicate the importance of these residues in the conformational constraints on all-trans-retinol that are necessary to form a catalytically competent complex.


FOOTNOTES

*
This work was supported by National Institute on Alcohol Abuse and Alcoholism Grants R37-AA02342, R37-AA07117, K21-AA00148, and K21-AA00150. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U09623[GenBank].

§
To whom correspondence should be sent: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., MS 405, Indianapolis, IN 46202-5122. Tel.: 317-274-7211; Fax: 317-274-4686.

(^1)
The abbreviations used are: ADH, alcohol dehydrogenase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).


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