X-ray Structure of Human Class IV sigma sigma Alcohol Dehydrogenase
STRUCTURAL BASIS FOR SUBSTRATE SPECIFICITY*

(Received for publication, February 18, 1997, and in revised form, May 7, 1997)

Peiguang Xie , Stephen H. Parsons , David C. Speckhard Dagger , William F. Bosron and Thomas D. Hurley

From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202 and the Dagger  Department of Chemistry, Loras College, Dubuque, Iowa 52001

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The structural determinants of substrate recognition in the human class IV, or sigma sigma , alcohol dehydrogenase (ADH) isoenzyme were examined through x-ray crystallography and site-directed mutagenesis. The crystal structure of sigma sigma ADH complexed with NAD+ and acetate was solved to 3-Å resolution. The human beta 1beta 1 and sigma sigma ADH isoenzymes share 69% sequence identity and exhibit dramatically different kinetic properties. Differences in the amino acids at positions 57, 116, 141, 309, and 317 create a different topology within the sigma sigma substrate-binding pocket, relative to the beta 1beta 1 isoenzyme. The nicotinamide ring of the NAD(H) molecule, in the sigma sigma structure, appears to be twisted relative to its position in the beta 1beta 1 isoenzyme. In conjunction with movements of Thr-48 and Phe-93, this twist widens the substrate pocket in the vicinity of the catalytic zinc and may contribute to this isoenzyme's high Km for small substrates. The presence of Met-57, Met-141, and Phe-309 narrow the middle region of the sigma sigma substrate pocket and may explain the substantially decreased Km values with increased chain length of substrates in sigma sigma ADH. The kinetic properties of a mutant sigma sigma enzyme (sigma 309L317A) suggest that widening the middle region of the substrate pocket increases Km by weakening the interactions between the enzyme and smaller substrates while not affecting the binding of longer alcohols, such as hexanol and retinol.


INTRODUCTION

Human alcohol dehydrogenase (ADH)1 isoenzymes are NAD+-dependent, zinc metalloenzymes that catalyze the reversible oxidation of alcohols to aldehydes. The ADH system is the major pathway for the metabolism of beverage ethanol as well as biological important alcohols or aldehydes like retinol, 3beta -hydroxysteroids, omega -hydroxy fatty acids, and 4-hydroxynonenal (1-3). Each isoenzyme in the ADH family is a dimer comprised of two 40-kDa subunits. The individual subunits are comprised of two domains, a catalytic domain and a coenzyme-binding domain (4). Seven human ADH genes (ADH1-ADH7) have been identified (1, 5). The ADH1-ADH5 genes encode the alpha , beta , gamma , pi , and chi  subunits, respectively. The protein product of the ADH6 gene has not been identified in vivo. The sigma  subunit is encoded by ADH7. Polymorphism occurs at both the ADH2 (beta 1, beta 2, and beta 3) and ADH3 (gamma 1 and gamma 2) loci (6), such that nine distinct human ADH subunits have been identified. ADH isoenzymes have been assigned to five distinct classes based on their amino acid sequences as well as their electrophoretic and enzymatic properties (7). The human alpha alpha , beta beta , and gamma gamma isoenzymes comprise class I, and the pi pi , chi chi , sigma sigma , and ADH6 comprise classes II, III, IV, and V, respectively. All ADH isoenzymes are expressed in the liver except for sigma sigma ADH, which is primarily localized in epithelial tissue, such as the stomach mucosa (8, 9).

The three-dimensional structures of horse and human class I ADHs have been solved by x-ray crystallography (4, 10-12). Recently, the structure of human class III ADH was reported (13), as well as the structure of a cod liver ADH isoenzyme (14). Thus, an increasingly diverse structural data base exists from which information concerning the determinants of substrate recognition can be obtained by comparing the structures and kinetic properties of ADH isoenzymes. Important amino acids within the substrate-binding site directly affect the substrate specificity of the human ADH isoenzymes (Table I). For instance, gamma 1gamma 1 ADH, which has a Ser at residue 48, is the only human isoenzyme able to bind and oxidize 3beta -hydroxysteroids (15). Amino acid substitutions within the loops comprised of residues 55-61 and of residues 113-121 in chi chi ADH cause these loops to adopt new conformations and contribute to the enzyme's inability to be saturated with ethanol (13). Mutagenesis studies on the beta 1beta 1 isoenzyme indicate that residues 93 and 94 account for the increased catalytic efficiency toward secondary alcohols exhibited by alpha alpha , relative to beta 1beta 1 (16). Residue 116, located at the entrance to the substrate pocket, also affects the Km for alcohols by acting as a bottleneck (17).

Table I. Genetic relationship among human alcohol dehydrogenases and amino acid residues involved in substrate binding


Subunit Gene Sequence identity to beta 1beta 1 Amino acid residues in the substrate-binding site
48 57 93 94 116 140 141 294 306 309

%
 alpha alpha ADH1 94 T M A I V F M V M L
 beta 1beta 1 ADH2 100 T L F T L F L V M L
 gamma 1gamma 1 ADH3 95 S L F T L F V V M L
 pi pi ADH4 60 T F Y A N F F V E I
 chi chi ADH5 62 T D Y I V Y M V F V
ADH6 ADH6 63 T H F L Q F G V Q F
 sigma sigma ADH7 69 T M F L I F M V M F

Among the human class I enzymes, beta 1beta 1 exhibits the lowest Km for ethanol (18), 120- and 7-fold lower than alpha alpha and gamma 1gamma 1, respectively. From ethanol to hexanol, the catalytic efficiency (Vmax/Km) of the alpha alpha isoenzyme increases 400-fold, while it increases just slightly more than 2-fold for the beta 1beta 1 isoenzyme (18). Compared with class I beta 1beta 1 ADH, class IV sigma sigma ADH also exhibits very different substrate binding characteristics. The Km for ethanol exhibited by sigma sigma ADH is 215-fold higher than that for hexanol (9). In addition, sigma sigma ADH exhibits the highest catalytic efficiency for the oxidation of all-trans-retinol to all-trans-retinal among the known human ADH isoenzymes (19). The production of all-trans-retinal from retinol is thought to be the rate-controlling step for the production of all-trans-retinoic acid (20), an important regulator of gene expression during embryonic development (21). The pathway of retinoic acid biosynthesis involves retinoid-binding proteins, which may provide a mechanism to discriminate specific dehydrogenases from nonspecific dehydrogenases (22). Retinol, in vivo, is bound to the cellular retinol-binding protein (CRBP). Evidence has been presented showing that holo-CRBP serves as substrate for microsomal dehydrogenases (22) and that CRBP may then transfer retinal to the cytosolic retinal dehydrogenase for oxidation to retinoic acid. Complete dependence on the CRBP pathway for retinoic acid production may deny accessibility of retinol to sigma sigma ADH in vivo. However, retinoic acid synthesis during embryogenesis was reported to correlate spatiotemporally with the expression of class IV ADH gene (23). It was proposed that competitive inhibition by ethanol consumed during pregnancy can reduce retinoic acid synthesis and may contribute to the development of fetal alcohol syndrome (24, 25).

In this paper, we examine the structural basis for substrate recognition in sigma sigma ADH through x-ray crystallography and site-directed mutagenesis. By comparing the structures of the known human ADH substrate-binding sites, it may be possible to gain a more complete understanding of their roles in the metabolism of endogenous and exogenous alcohols.


EXPERIMENTAL PROCEDURES

Protein Purification and Crystallization

The cDNA for the sigma  subunit (5) in M13 was subcloned into the vector pKK223-3 (Pharmacia Biotech Inc.) by site-directed mutagenesis using a commercial kit (Amersham Corp.) and expressed in Escherichia coli as described for beta 1beta 1 ADH (10). The lysate was first mixed with DEAE-cellulose (Whatman, Maidstone, UK) in 50 mM Tris, pH 8.8, at 4 °C, 1 mM benzamidine, 2 mM dithiothreitol. The unbound proteins were eluted in a batch procedure and then were buffer exchanged into 7 mM sodium phosphate, pH 6.4, 1 mM DTT using the Minitan apparatus (Millipore, Bedford, MA) and loaded onto a 5- × 15-cm S-Sepharose column. The protein was eluted with a linear sodium phosphate gradient from 7 to 65 mM. The enzyme was dialyzed into 10 mM sodium phosphate, pH 6.4, 1 mM DTT and applied to a 2.5- × 10-cm Affi-Gel Blue column (Bio-Rad). The enzyme was then eluted with a linear gradient from 10 mM sodium phosphate, pH 6.4, to 100 mM Tris, pH 8.8, 1 mM DTT. The purified sigma sigma ADH was dialyzed into 10 mM HEPES, pH 7.0, 1 mM DTT and concentrated with a Microcon 30 concentrator (Amicon, Beverly, MA) before crystallization. The sitting drop method was employed to crystallize the protein. Typically 2 µl of an 8 mg/ml sigma sigma ADH solution was mixed with 2 µl of the precipitant solution in the drop. The optimized crystallization conditions for sigma sigma ADH complexed with NAD+ were 100 mM cacodylate, pH 6.5, 50-100 mM zinc acetate, 7.5 mM NAD+, and 18% (w/v) polyethylene glycol 6000. The crystals formed as flat parallelepipeds overnight and grew to maximal size in 1 or 2 more days.

Mutagenesis and Kinetic Studies

Single-stranded sigma sigma cDNA in the M13HinEco1 vector (26) was used as the template for site-directed mutagenesis. A single oligonucleotide, 45 bases in length, was used to mutate residues Phe-309 and Cys-317 to Leu and Ala, respectively. Following identification of the correct mutant clone by DNA sequencing, the mutant cDNA was subcloned into pKK223-3 and completely sequenced, prior to expression, to ensure that no unwanted mutations were present in the cDNA sequence. The mutant enzyme, sigma 309L317A, was expressed and purified using the same procedure as described for the wild-type enzyme. The kinetic measurements were evaluated at 25 °C in 100 mM sodium phosphate, pH 7.5, on a Beckman DU-640 spectrophotometer. Enzyme activity was monitored by following the production of NADH at 340 nm using an extinction coefficient of 6.22 mM-1 cm-1. Vmax values were converted to turnover numbers assuming a molecular mass of 40 kDa per subunit. The Km values for substrates were determined at a fixed NAD+ concentration of 2.5 mM, except those for 1-butanol which were determined both at 2.5 mM NAD+ and by co-variation of NAD+ and 1-butanol. All kinetic experiments were evaluated using the kinetic programs of Cleland (27). All reported values are expressed as the means of at least three separate experiments with their associated standard deviations.

X-ray Diffraction Data Collection

X-ray diffraction data were collected to 3 Å. Higher resolution data were observed initially (2.6 Å), but severe radiation decay and the inability to flash-cool these crystals prevented collection of the higher resolution data. Four crystals (approximate dimensions, 0.3 × 0.15 × 0.07 mm3) were used to collect the native data set at room temperature on a Rigaku 200HB rotating anode generator equipped with an RAXIS IIC image plate area detector with a crystal-to-detector distance of 145 mm. The data collection statistics are listed in Table II. All crystals exhibited radiation decay and were replaced every 12 h. The diffraction data were indexed, merged, and scaled using the RAXIS IIC data processing software (38).

Table II. Data collection and molecular refinement statistics

Rmerge = Sigma hkl|Ii - In|/Sigma In where Ii is an observed intensity and In is the average of the observed equivalents. Rwork = Sigma hklpar-bars Fobs|-|Fcalpar-bars /Sigma hkl|Fobs| where |Fobs| and |Fcal| are the observed and calculated structure factor amplitudes of a reflection hkl, respectively. Rfree is the same as Rwork except that the summation is over the portion of data (7%) that is not included in the refinement.

Space group P21 (monoclinic)
Cell parameters a,b,c (Å), alpha , beta , gamma  (°) 86.3, 94.7, 121.7, 90.0,   100.0, 90.0 
Maximal resolution (Å) 3.0
Number of observations (35-3 Å) 79,865
Number of unique reflections 34,754
Completeness to 3.0 Å 87%
Completeness of the outer shell (3.5-3.0 Å) 83.5%
Rmerge (%) 12
Rwork/Rfree (%)
  Rigid body refinement (8-3.5 Å) 37.3/37.7
  Atomic position refinement (8-3 Å) 22.5/30.5
Luzatti estimate of coordinate error (Å) 0.33
r.m.s. deviation from ideal bond length (Å) 0.01
r.m.s. deviation from ideal bond angle (°) 2.0

Molecular Replacement and Crystallographic Refinement

The structure was solved by molecular replacement using the program package AMoRe (28) and the data between 15.0 and 4.0 Å. The beta 1beta 1 ADH ternary complex dimer with NAD+ and the inhibitor 4-iodopyrazole (Protein Data Bank code 1DEH (13)) served as the search model for these calculations. The correlation coefficients for the top two rotation solutions were 25.7 and 19.7, respectively. After the positions for the two dimers in the asymmetric unit were found, the starting model possessed a correlation coefficient of 54.7 and an R value of 39.9%. All subsequent model refinement was performed using the program package X-PLOR (version 3.1) (29). Rigid-body refinement of the initial model structure with the data between 8.0 and 3.5 Å brought Rwork from 40.2 to 37.7% and Rfree from 40.5 to 37.3%. The atomic positions were refined to 3 Å using the positional refinement protocol in X-PLOR (30) and an overall temperature factor of 25 Å2. The resulting structure was inspected using 2Fo - Fc and Fo - Fc maps in CHAIN (31). Amino acid substitutions were introduced as their positions were identified during refinement. Additional solvent zinc cations and solvent acetate molecules were added when strong positive Fo - Fc electron density indicated their presence. In the last refinement procedure, an overall temperature factor for each subunit was refined, and the non-crystallographic symmetry restraints were removed. The final model possesses an average r.m.s.d. of 0.2 Å for the main chain atoms in the four subunits of the asymmetric unit. Calpha alignments between sigma sigma and beta 1beta 1 isoenzymes were performed using LSQKAB (32) in CCP4 (1994) suite and displayed using QUANTA (Molecular Simulations Inc., Burlington, MA).

Reagents

NAD+, grade I and DTT were purchased from Boehringer Mannhein, and PEG 6000 was purchased from Hampton Research; ethanol was purchased from Midwest Grain (Pekin, IL). All other reagents were from Sigma and were of the highest grade available.


RESULTS

Structure Determination

The structure of the human class IV, or sigma sigma , ADH isoenzyme was solved to 3.0 Å by molecular replacement using the 2.2-Å structure of the class I human beta 1beta 1 isoenzyme (12) as the starting model. The final refined structure possesses an Rwork of 22.5% with an Rfree of 30.5% (Table II). The stereochemistry of this model was inspected using the program package PROCHECK (33). The Ramachadram plot showed that 98.6%, or 1471, of the 1492 residues were in the preferred and allowed regions, and 1.4%, or 21, of the residues were in the generously allowed region. No non-glycine residues were found in the disallowed region. Due to the presence of zinc acetate in the mother liquor, 8 solvent zinc cations and 10 acetate ions were identified as bound to the enzyme. There was an acetate ion present in the substrate pocket in all four subunits.

Kinetics of the sigma sigma ADH Mutant

A sigma sigma ADH mutant, sigma 309L317A, was prepared by site-directed mutagenesis. Two residues in the sigma sigma isoenzyme, Phe-309 and Cys-317, were mutated to Leu and Ala, respectively. The choice of these two positions for mutagenesis was based on their unique characteristics compared with class I enzymes (Table I). Residue 309 is in the substrate-binding pocket, and residue 317 is behind the nicotinamide ring of NAD+. The substrate specificity of this mutant was studied and compared with wild-type sigma sigma ADH (Table III). Mutations at these two residues dramatically increase the Km values toward small alcohol substrates. For instance, the mutant enzyme exhibits a Km for ethanol that is 100-fold higher than the wild-type sigma sigma ADH. Interestingly, the Km values for substrates with five or more carbons are less affected. The Km values for hexanol and retinol are essentially unaffected by these mutations. As the chain length for straight alcohols increases, the Vmax/Km of the mutant enzyme increases to a greater extent than does the wild-type enzyme. The Km value for NAD+ was approximately 2 times higher than the wild-type enzyme, whereas the Ki(NAD+) value obtained from experiments varying both 1-butanol and NAD+ concentrations was identical with the wild-type enzyme (0.75 ± 0.03 mM).

Table III. Kinetic constants of human sigma sigma , sigma 309L317A mutant, and beta 1beta 1 ADHs at 25 °C

The kinetic constants were determined at pH 7.4 or 7.5 for recombinant ADHs, except for the Km for NAD+ of sigma sigma ADH, which was determined from the stomach isolated enzyme. The data for beta 1beta 1 and wild-type sigma sigma are from Refs. 5, 9, 16, and 19.

Km
Vmax/Km
 beta 1beta 1 Mutant  sigma sigma  beta 1beta 1 Mutant  sigma sigma

mM min-1 mM-1
Ethanol 0.05 2920  ± 370 28  80 1.1  ± 0.2   65
1-Propyl alcohol 0.019 330  ± 55 1.39 160 3.2  ± 0.6  570
1-Butanol 0.012 51  ± 8 0.79 240 33  ± 6 2640
1-Pentanol 0.029 1.9  ± 0.4 0.28 190 510  ± 75 3400
1-Hexanol 0.022 0.18  ± 0.02 0.13 200 6300  ± 780 9000
All-trans-retinol 0.045 0.049  ± 0.011 0.031  20 250  ± 35 1900
NAD+ 0.015 0.529  ± 0.015 0.26 270a 5590a 7080a

a Calculated for ethanol oxidation.

Structural Comparison with Other ADH Structures

An alignment of the Calpha atoms, excluding residues 113-120 and 244-262, in the dimeric sigma sigma and beta 1beta 1 isoenzymes gives a r.m.s.d. of 0.60 Å. Alignment of the individual domains within each subunit yields similar results, with r.m.s.d. values of 0.49 Å for the catalytic domain and 0.61 Å for the coenzyme-binding domain. Calpha alignment of sigma sigma ADH with horse and other human ADHs shows that its structure is most similar to the human and horse liver class I ADH isoenzymes. Unlike the recently reported structures of the human class III chi chi isoenzyme (13) and the ADH isoenzyme from cod liver (14), both of which exhibited semi-open domain structures, the human sigma sigma isoenzyme exhibits a fully closed conformation of the catalytic and coenzyme-binding domains when NAD(H) is bound. The alignments reveal that, relative to beta 1, there are two major structural differences in each domain of the sigma  subunit (Fig. 1). In the coenzyme binding domain, the largest difference occurs at the C terminus of an alpha -helix comprised of residues 251-258 and the following turn. This structural change results from the substitutions of the two Gly residues at positions 260 and 261 in beta 1beta 1 by two Asn residues in sigma sigma ADH. The main chain conformations of Gly-260 and -261 in the beta 1beta 1 isoenzyme are incompatible with the presence of side chains, thus the main chain path must shift dramatically to accommodate Asn residues at positions 260 and 261. Relative to beta 1beta 1 ADH, the Calpha atoms of residues 244-262 in sigma sigma ADH shift, on average, by 1.8 Å, with a maximum of 4.8 Å at residue 261. The other structural difference in the coenzyme-binding domain involves residues 297-309, which are located at the dimer interface and within the substrate-binding pocket of the neighboring subunit. One structural difference in the catalytic domain involves residues 17-25 and is not likely to affect enzymatic activity or subunit interactions. The other structural difference within the catalytic domain is due to the deletion of residue 117, which shortens the loop at the entrance to the substrate-binding pocket in sigma sigma ADH. Calpha alignment between sigma sigma and chi chi , with each domain aligned separately and then the results combined, reveals that in addition to those differences between sigma sigma and beta 1beta 1, sigma sigma and chi chi differ substantially at both N and C termini (Fig. 1). Moreover, great differences exist at residues 55-61 and 112-120. Both regions adopt new conformations in chi chi ADH and contribute to a larger active site in chi chi ADH (13).


Fig. 1. Comparison of sigma sigma , chi chi , and beta 1beta 1 dimers by alignment of their Calpha atoms. All Calpha atoms were used in the alignment. The result of the alignment using the CD dimer of sigma sigma ADH is shown. The other dimer of sigma sigma ADH in the asymmetric unit gives similar results.
[View Larger Version of this Image (27K GIF file)]

Our structural comparisons also reveal that there is no evidence that the interaction between coenzyme and residue 223 is weakened in sigma sigma ADH, as was suggested by a modeling study (34). In fact, the hydrogen-bonding distances between adenosine ribose oxygens and gamma  oxygens of Asp-223 are within the range of 2.6-2.7 Å in both sigma sigma and beta 1beta 1 ADHs.


DISCUSSION

The alcohol binding pocket is an extension of the coenzyme binding site (4) and is fully formed only after coenzyme binding has occurred. In sigma sigma ADH, the substrate pocket is a cylinder having dimensions of approximately 16 by 7 by 6 Å. The substrate specificity for this enzyme is determined by surface complementarity between the enzyme and the substrates throughout this cylinder. Changes in the Km values are related to the effective concentration of the ES complex. Mutations can affect Km by changing the ratio of productive versus non-productive encounters with the enzyme. In ADH, these changes are brought about either through steric exclusion (preventing productive binding), as seen for the binding of secondary alcohols to beta 1beta 1 ADH (16), or by changing the number of non-productive conformations permitted by altering the accessible volume of the active site (13). The inner part of the alcohol site (near the catalytic zinc) includes residues 48 and 93 and the nicotinamide ring of NAD+. sigma sigma ADH has a Km value for ethanol that is 560-fold higher than beta 1beta 1 ADH (Table III). One possible cause for this difference may be the substitution of Cys for Ala-317 near the nicotinamide ring. To accommodate its longer side chain in sigma sigma ADH, the main chain atoms of residue 317 move ~1 Å away from Thr-186, toward the carboxamide group relative to beta 1beta 1 ADH (Fig. 2). To avoid unfavorable contacts with the Cys-317 carbonyl oxygen, the plane of nicotinamide ring appears to twist in sigma sigma ADH, relative to its position in the beta 1beta 1 isoenzyme (Figs. 2 and 3), This twist creates more space between the nicotinamide ring and the catalytic zinc in sigma sigma ADH (Fig. 3). In addition to these changes, Thr-48 and Phe-93 also shift away from the catalytic zinc. The distance between the Calpha atoms of these two residues is 0.9 Å longer in sigma sigma ADH. Consequently, smaller substrates, such as ethanol, are not as conformationally constrained in this active site as in the beta 1beta 1 ADH. Thus, a higher concentration of ethanol is required to produce an equivalent number of productively bound conformations.


Fig. 2. The influence of the substitution of Ala-317 right-arrow-Cys in sigma sigma on the conformation of the nicotinamide ring of NAD+. The residues in sigma sigma ADH are shown using the thicker line. The electron density map is from the sigma  structure and contoured at 1.0 S.D. of the map.
[View Larger Version of this Image (40K GIF file)]


Fig. 3. Comparison of the active site in sigma sigma and beta 1beta 1 ADH isoenzymes. a, superposition of the active site between two isoenzymes. The alignment is performed on the Calpha of the coenzyme binding domain excluding residues 244-262. b, the accessible surfaces of the substrate pocket in sigma sigma (in dark dots) and beta 1beta 1 ADHs. This orientation is approximately perpendicular to that in a, with the catalytic zinc at the left and the entrance at the right. A hexanol molecule was introduced artificially to indicate the approximate size of the pocket. The approximate side chain positions of two residues are also indicated.
[View Larger Version of this Image (39K GIF file)]

The structural differences near the catalytic zinc in these two isoenzymes may also explain the weak binding of the inhibitor pyrazole to sigma sigma ADH (Ki values of 0.60 µM for beta 1beta 1 and 350 µM for sigma sigma at pH 7.5 (5, 12)). Pyrazole and its 4-substituted derivatives competitively inhibit the binding of alcohol substrates through the formation of a tight enzyme·NAD+·inhibitor complex (35), in which pyrazole nitrogens interact with both zinc and NAD+. We speculate the bond between the pyrazole nitrogen atom and the C-4 atom on the nicotinamide ring may be distorted due to the twist of the nicotinamide ring in sigma sigma ADH. In fact, if the active sites of the sigma sigma and beta 1beta 1 structures are aligned and the position of 4-iodopyrazole in the beta 1beta 1 active site structure is used to examine the geometric constraints on pyrazole binding to the sigma sigma structure, the corresponding angle between the C-4-N-1 and the N-1-N-2 bond is 133°, while it is close to 120° in the beta 1beta 1 structure, corresponding to a low energy, stable complex. This angular difference would undoubtedly represent a higher energy conformation and could account for up to 2.7 kcal/mol of the observed difference (3.8 kcal/mol) using a harmonic potential with a force constant of 0.27 kcal/(mol·degree) (36). In addition, the increased distances between the N-1 of pyrazole and the C-4 of the nicotinamide ring (by 0.3 Å) and between residues 48 and 93, where pyrazole is held, could contribute to lowering the affinity for 4-methylpyrazole.

The middle region of the substrate pocket, which plays an important role in the interactions with the aliphatic tail of longer substrates, such as butanol and pentanol, includes residues 57, 141, 294, and 309. Like many other ADH isoenzymes, sigma sigma ADH exhibits Km values for primary straight chain alcohols, which decrease with increasing chain length, whereas the Vmax values remain relatively constant. Thus, the catalytic efficiency (Vmax/Km) increases with increasing chain length. For example, the Vmax/Km for hexanol is 138-fold higher than that for ethanol in sigma sigma ADH (Table III). In contrast, the Vmax/Km values for beta 1beta 1 ADH vary only 2- to 3-fold for substrates from ethanol to hexanol. These different characteristics can be explained by differences in the amino acids within the middle region of the substrate pocket. The key amino acids within this region are residues 57, 141, and 309. beta 1beta 1 ADH possesses Leu residues at all these positions, and their side chains do not appear to create new productive interactions as substrates get longer (Table III). The presence of Phe at position 309, Met at position 141, and Met at position 57, in sigma sigma ADH, narrows the middle region of the substrate pocket compared with beta 1beta 1 (Fig. 3). The twist on the nicotinamide ring also contributes to a shift in Phe-309, to avoid unfavorable contact with NAD(H), which further narrows the channel leading to the catalytic zinc. Although this narrowing does not appear to directly aid the binding of ethanol, it can explain the decreased Km values for propanol, butanol, and pentanol in the sigma sigma isoenzyme relative to beta 1beta 1 ADH.

Our modeling indicated that the side chain of residues 57 and 309 would interact with the substrates at the carbon 4, 5, and 6 positions, whereas the side chain of residue 141 interacts with carbon 3 and 4. In the sigma 309L317A mutant, the Km values for propanol, butanol, and pentanol are increased by 240-, 60-, and 7-fold, confirming the role of residues 309 and 317 in stabilizing the binding of these substrates. Moreover, the increase in Vmax/Km versus the chain length of the substrates is greater for the mutant than for the wild-type enzyme. This behavior in the mutant enzyme is due to a much lower Vmax/Km for small substrates, since the Vmax/Km values for hexanol approach those of the wild-type enzyme. The substitutions in the mutant enzyme thus appear to further widen the substrate-binding site, relative to sigma sigma ADH, resulting in a greater number of permissible non-productive ES complexes. These changes in the mutant only affect substrates where binding is dependent on the local topology, such as ethanol, but do not significantly affect the catalytic efficiency toward hexanol or retinol.

The outer part of the substrate-binding pocket is exposed to solvent and includes the loop comprised of residues 114-120 and residue 306 from the other subunit within the dimer. The deletion of residue 117 in sigma sigma ADH shortens the loop comprised of residues 114-120 (Fig. 3) and widens the entrance to the substrate-binding site. In beta 1beta 1 ADH, Leu-116 appears to function as a door, opening to allow substrates in or out, but then closing to help keep in bound substrate (17). Consistent with this function, its side chain was found to occupy different conformations in binary and ternary complexes (10-12). In sigma sigma ADH, the shift in the position of residue 116 due to the deletion of residue 117 does not permit its side chain to function in this manner, leaving an open substrate-binding site (Fig. 3). Widening of the bottleneck at position 116 in beta 1beta 1 ADH by mutagenesis dramatically increased the apparent Km values for primary and secondary alcohols (17). Consistent with these observations, sigma sigma ADH has higher Km values than the beta 1beta 1 isoenzyme for straight chain alcohols and very poor efficiency toward all secondary alcohols (5). The Km for hexanol and retinol are virtually identical in the wild-type and mutant enzymes. With the enlarged entrance to the substrate pocket in the sigma sigma isoenzyme, the structure of the middle and inner regions of the substrate pocket would seem to be best suited for the oxidation of long chain aliphatic alcohols, such as omega -hydroxy fatty acids, farnesyl alcohols, and retinol. To examine long chain alcohol binding, all-trans-retinol was docked into the active site using program AUTODOCK (37). The results of this simulation confirm our previous results based on modeling studies (5), the beta -ionone ring of retinol binds at the widened entrance of the substrate-binding pocket, such that an extended conformation of retinol can be adopted. Thus, the ability to bind retinol in a more extended and, presumably, lower energy conformation in sigma sigma ADH could account for its higher catalytic efficiency.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants K21-AA00150, R29-AA10399, R37-07117, and P50-07611.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1AGN) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.


Dagger    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5122. Tel.: 317-278-2008; Fax: 317-274-4686; E-mail: hurley{at}biochem6.iupui.edu.
1   The abbreviations used are: ADH, alcohol dehydrogenase; DTT, dithiothreitol; r.m.s.d., root-mean-square deviation; CRBP, cellular retinol-binding protein.

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