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FEMS Yeast Research 2 (2002) 481^494 www.fems-microbiology.org MiniReview The three zinc-containing alcohol dehydrogenases from baker’s yeast, Saccharomyces cerevisiae Vladimir Leskovac a a; , Svetlana Trivic¤ b , Draginja Peric›in a Faculty of Technology, The University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Yugoslavia b Faculty of Science, The University of Novi Sad, 21000 Novi Sad, Yugoslavia Received 7 February 2002; received in revised form 1 August 2002 ; accepted 2 August 2002 First published online 18 September 2002 Abstract This review is a summary of our current knowledge of the structure, function and mechanism of action of the three zinc-containing alcohol dehydrogenases, YADH-1, YADH-2 and YADH-3, in baker’s yeast, Saccharomyces cerevisiae. The opening section deals with the substrate specificity of the enzymes, covering the steady-state kinetic data for its most known substrates. In the following sections, the kinetic mechanism for this enzyme is reported, along with the values of all rate constants in the mechanism. The complete primary structures of the three isoenzymes of YADH are given, and the model of the 3D structure of the active site is presented. All known artificial mutations in the primary structure of the YADH are covered in full and described in detail. Further, the chemical mechanism of action for YADH is presented along with the complement of steady-state and ligand-binding data supporting this mechanism. Finally, the bio-organic chemistry of the hydride-transfer reactions catalyzed by the enzyme is covered: this chemistry explains the narrow substrate specificity and the enantioselectivity of the yeast enzyme. 5 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Yeast alcohol dehydrogenase; Alcohol dehydrogenase ; Saccharomyces cerevisiae 1. Introduction Yeast alcohol dehydrogenase (EC 1.1.1.1) is a member of a large family of zinc-containing alcohol dehydrogenases. The primary structures of 47 members of this family have been determined and aligned, and an evolutionary tree has been constructed, assuming a divergent evolution from a common ancestral gene [1]. In this way, it was possible to identify four divergent groups of alcohol dehydrogenases in this family: vertebrates, plants, eukaryotic microorganisms and prokaryotic bacteria. Baker’s yeast (Saccharomyces cerevisiae), a member of the third group, has three isoenzymes of alcohol dehydrogenase : YADH-1, YADH-2, and YADH-3. YADH-1 is the constitutive form that is expressed during anaerobic fermentation [2]. YADH-2 is another cytoplasmic form, which is repressed by glucose [3], and YADH-3 is found in the mitochondria [4]. YADH-1 accounts for the major part of alcohol dehydrogenase activity in growing baker’s yeast. The structure, function and mechanism of action of yeast alcohol dehydrogenase have been reviewed three decades ago [5,6]. The purpose of this article is to update the subject and to review novel data on the structure, function and mechanism of action of the isoenzyme YADH-1; this isoenzyme will be abbreviated as YADH throughout the text. The steady-state kinetic constants are presented in the nomenclature of Cleland [7]. 2. Isoenzymes of YADH * Corresponding author. Fax: +381 (21) 350 122. E-mail address : [email protected] (V. Leskovac). Abbreviations : YADH, yeast alcohol dehydrogenase, isoenzyme YADH-1; NDMA, p-nitroso-N,N-dimethylaniline ; AA, acetamide; Az, sodium azide; DACA, N,N-dimethylamino-trans-cinnamaldehyde Yeast alcohol dehydrogenase was one of the ¢rst enzymes to be puri¢ed and isolated [8]. If the steady-state kinetic properties of the ADH isoenzymes are compared, a large degree of similarity is detected. Table 1 shows the steady-state kinetic constants for the three isoenzymes of YADH, isolated from baker’s yeast. 1567-1356 / 02 / $22.00 5 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 5 7 - 5 FEMSYR 1513 11-11-02 482 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 Table 1 Steady-state kinetic constants of yeast ADH isoenzymes with ethanol and acetaldehyde as substrates, at pH 7.3, 30‡Ca Constant Unit YADH-1 YADH-2 YADH-3 V1 KA KB V1 /KB V2 KQ KP V2 /KP s31 WM mM mM31 s31 s31 WM mM mM31 s31 340 170 17 20 1700 110 1.1 1540 130 110 0.81 160 1040 50 0.09 11 550 450 240 12 37.5 2100 70 0.44 4770 a At neutral pH, the equilibrium is shifted far to the left (Table 2). Substrate speci¢city of YADH is restricted to primary unbranched aliphatic alcohols, and any branching in the side chain diminishes the activity of the enzyme and lowers its e⁄ciency. In addition, the enzyme also shows activity towards secondary alcohols. Table 2 presents the steadystate kinetic constants for various alcoholic substrates and Table 3 shows the steady-state constants for various carbonyl substrates of the yeast enzyme. Ethanol is by far the best substrate of the yeast enzyme. Methanol is a very poor substrate of YADH; the methanol activity of the enzyme at pH 8.8 is only 0.07% of its ethanol activity under identical conditions. The enzyme is able to oxidize methanol by NADþ to formaldehyde and NADH, but the enzymatic reaction is very complex due to interference of numerous side reactions [18]. Allyl and cinnamyl alcohol are, however, excellent substrates; kinetic constants for the latter alcohol are: V1 = 133 s31 and V1 /KB = 29 mM31 s31 , at pH 8.2, 25‡C [19]. (S)-(+)-Butan-2-ol is a much better substrate than (R)-(3)-butan-2-ol (V1 = 1.0 and 0.05 s31 , and V1 /KB = 18 and 0.8 M31 s31 , respectively, at pH 7.3, 30‡C) [20]. 4-Methyl-1-pentanol (V1 = 7 s31 , pH 8.2) is a much better substrate than 2-methyl-1-propanol (V1 = 0.2 s31 , pH 7.3) or 3-methyl-1-butanol (V1 = 0.3 s31 , pH 8.2) [19,20]. It was reported that glycerol, glyceraldehyde and acetol are poor substrates of YADH [21], whereas benzyl alcohol and benzaldehyde are extremely poor substrates of this enzyme [20,22]. It has also been reported that p-chlorobenzyl alcohol and p-methoxybenzyl alcohol are slowly oxidized by NADþ in the presence of YADH [23]. 2-Chloroethanol, 2-£uoroethanol, 2,2,2-tri£uoroethanol, propargyl alcohol, glycidol and polyethylene glycol are no substrates of the yeast enzyme [24]. Calculated from the data of Ganzhorn et al. [9]. It is evident that YADH-1 and YADH-3 have very similar kinetic characteristics, while YADH-2 has a much higher substrate speci¢city for ethanol (V1 /KB ) and acetaldehyde (V2 /KP ), and much lower Michaelis constants with ethanol (KB ) and acetaldehyde (KP ). Recently, the kinetic characterization of YADH-1 and YADH-2 has been extended by measuring their speci¢city constants (V1 /KB ) for a number of long-chain alcohols and diols. It was found that for all alcohols, normalized rates with YADH-2 were about three-fold faster than with YADH-1 [10]. 3. Substrate speci¢city Yeast alcohol dehydrogenase catalyzes the following reversible redox reaction [5]: ð1Þ Table 2 Steady-state kinetic constants for the oxidation of various alcohols at neutral pH Constant Unit Ethanola Propan1-ola Butan1-ola Hexan1-olb Decan1-olb Propan2-olc (S)-(+)-Butan2-olc Allyl alcohold Ethyleneglycold Trise V1 KA KiA KB V1 /KA V1 /KB 0.0001 V1 KiA /KA Keq f s31 WM WM mM mM31 s31 mM31 s31 454 109 325 21.7 4165 20.9 67 150 235 29.2 447 22.9 25 250 160 32 100 0.78 15.4 169 152 3.2 91 4.8 14.4 200 190 0.1 72 144 7 597 378 117 11.7 0.06 0.9 376 398 35 2.4 0.026 546 520 730 14.6 1058 37.5 7.0 370 550 444 19.2 0.016 0.5 698 842 6415 0.72 s31 ^ 1354 0.00019 105 ^ 16 0.00027 13.8 ^ 13.7 ^ 4.4 0.146 0.95 0.40 766 ^ 10.4 ^ 0.60 ^ a Calculated from the data of Calculated from the data of c Calculated from the data of d Calculated from the data of e Calculated from the data of f Keq = V1 KiQ KP /(V2 KiA KB ). b Dickinson and Monger [11], at pH 7.0, 25‡C. Scho«pp and Aurich [12], at pH 8.0, 25‡C. Trivic¤ and Leskovac [13], at pH 7.0, 25‡C. Trivic¤ and Leskovac [14], at pH 7.0, 25‡C. Chen and Huang [15], at pH 8.2, 25‡C. FEMSYR 1513 11-11-02 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 483 Table 3 Steady-state kinetic constants for the reduction of various carbonyl substrates at neutral pH Constant Unit Acetaldehydea Butyraldehydea Acetoneb Butan-2-one-b Chloroacetaldehydec NDMAd DACAe V2 KQ KiQ KP V2 /KQ V2 /KP V2 KiQ /KQ s31 WM WM mM mM31 s31 mM31 s31 s31 3850 96 12.5 0.9 40100 4280 501 3450 97 7 27.5 35570 125 249 9 43 17.5 477 209 0.019 3.66 0.7 38 15.2 285 18.4 0.0025 0.38 117 270 74 4 431 25.2 31.9 2.1 456 119 1.5 4.5 1.4 0.54 0.176 46 7.6 0.61 3.8 0.29 0.03 a Calculated Calculated c Calculated d Calculated e Calculated b from from from from from the data the data the data the data the data of of of of of Dickinson and Monger [11], at pH 7.0, 25‡C. Trivic¤ and Leskovac [13], at pH 7.0, 25‡C. Leskovac et al. [16], at pH 9.0, 25‡C. Trivic¤ et al. [17], at pH 8.9, 25‡C. Leskovac et al. [16], at pH 7.0, 25‡C. Yeast alcohol dehydrogenase catalyzes three essentially irreversible chemical reactions: ClWCH2 WCHO þ NADH þ Hþ ! ClWCH2 WCH2 OH þ NADþ ð2Þ ðCH3 Þ2 NWC6 H4 WNO þ 2NADH þ 2Hþ ! ðCH3 Þ2 NWC6 H4 WNH2 þ 2NADþ þ H2 O ð3Þ CH3 CHO þ NADþ þ H2 O ! CH3 COOH þ NADH þ Hþ ð4Þ Chloroacetaldehyde is an excellent substrate of YADH (Table 3), while 2-chloroethanol is not oxidized by NADþ , which makes the reaction 2 essentially irreversible [16]. p-Nitroso-N,N-dimethylaniline (NDMA) is readily reduced by NADH, in the presence of YADH (reaction 4); the primary product of this reaction, the corresponding hydroxylamine, is transformed into a quinonediimine compound by the loss of a molecule of water. The last compound is reduced non-enzymatically by NADH to p-amino-N,N-dimethylaniline [17,25]. YADH has a weak aldehyde dehydrogenase activity; it is able to catalyze an irreversible oxidation of acetaldehyde to acetic acid with NADþ , with an apparent kcat = 2.3 s31 and V/K = 34 M31 s31 , at pH 8.8, 22‡C [26]. Free acetaldehyde is a true substrate for alcohol dehydrogenase [27], and gem-diol is probably a true substrate for aldehyde dehydrogenase activity of YADH [26]. steady-state random mechanism on the alcohol side, and a steady-state ordered mechanism on the aldehyde side of the catalytic cycle, with primary aliphatic alcohols and aldehydes (Scheme 1). The mechanism in Scheme 1 is restricted to primary unbranched aliphatic alcohols and aldehydes, if the latter are present in lower concentrations [33]. The initial rate equation for this mechanism, in the forward direction and in the absence of products, is given by [37]: E0 X 1 1 ¼ þ þ V0 k9 k 5 k7 þ 1 Xk1 k14 k12 þ k13 A þ k11 B 1 W þ k1 k9 k13 k12 þ k13 A þ ðk13 =k1 Þk11 B A 1 Xk4 k12 þ k13 A 1 W þ þ k3 k 9 k12 þ k13 A þ ðk13 =k1 Þk11 B B þ 1 Xk4 k12 þ k13 A þ k11 B 1 W þ k3 k 9 k12 þ k13 A þ ðk13 =k1 Þk11 B AB ð5Þ where X = 1+k10 /k5 . The applicability of Eq. 5 to alcohol oxidation is readily apparent. Eq. 5 predicts that the monomolecular kinetic constant V1 and the bimolecular speci¢city constants V1 / KA and V1 /KB are dependent on the nature of substrate B; inspection of data in Table 2 shows that this is true for all primary unbranched alcohols. Also, Eq. 5 predicts that the inhibitory constant KiA is dependent on the nature of substrate B and, therefore, cannot be equal to the dissociation constant of the EWNADþ -complex ; Table 2 shows that this is true for all the above alcohols. In addition, a direct determination of Kþ E;NAD shows that it is not equal to 4. Steady-state kinetic mechanism Yeast alcohol dehydrogenase catalyzes the chemical reactions described by Eq. 1. Numerous investigations of the steady-state kinetic mechanism of the yeast enzyme have been conducted by several authors [9,11,28^36] ; they have led to the conclusion that the yeast enzyme follows the FEMSYR 1513 11-11-02 Scheme 1. 484 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 Table 4 Thermodynamics of the yeast alcohol dehydrogenase reaction, at pH 7.0, 25‡C [45] k1 k31 k32 k9 k5 k7 k2 k41 k42 k10 k6 k8 E3EA3EA3EAB3EPQ3EQ3E Rate constant vG0 (kJ/mol) Dissociation constant s k1 k2 k4 /k3 WM s31 WM k9 k10 k5 k6 k7 k8 s31 s31 s31 WM31 s31 s31 WM31 s31 31 31 a 7 X 0.2 2100 X 57 158 000 (11) (3900)a (^)a 3980 X 97 35 040 X 870 10 900 X 160 5.0 X 0.04 388 X 5 28.1 X 0.5 (4000)a (35 000)a (11 000)a (4.3)a (480)a (44)a k2 /k1 WM 300 320.08 k41 /k31 k42 /k32 k10 /k9 ^ WM 75b 2110c 8.75 10.70 315.26 5.37 k5 /k6 WM 2180 15.18 k7 /k8 WM 13.80 27.73 Total Keq = 0.000068d 23.64 23.7 a Data in parentheses are from the steady-state kinetic measurements of Dickinson and Dickenson [31], at pH 7.0, 25‡C. Taken from Northrop [46]. c Calculated from the equilibrium constant: k4 /k3 = (k41 /k31 )/(k42 /k32 ). d Calculated from the Haldane relationship : Keq = V1 KiQ Kp /(V2 KiA KB ). b KiA , in any case (Fig. 4). The initial-rate equation in the reverse direction, reduction of aldehydes, and in the absence of substrates of reaction, is given by the general expression for the steady-state ordered mechanism [38] : E0 Y 1 1 1 Yk5 þ k10 k7 ðYk5 þ k10 Þ þ ¼ þ þ þ þ V0 k6 k10 P k2 k4 k10 k8 Q k6 k8 k10 PQ ð6Þ where Y = 1+k9 /k4 . Eq. 6 satis¢es the results obtained for the reduction of acetaldehyde and butyraldehyde in predicting a linear reciprocal equation, in which the KiQ , V2 /KQ and V2 KiQ /KQ constants are independent of the nature of the aldehyde (Table 3). The kinetic mechanism in Scheme 1 is compatible with deuterium isotope e¡ects on maximal rates reported for ethanol, D V1 = 1.8, D V1 /KA = 1.8, and D V1 /KB = 3.2 [39], propan-1-ol, D V1 = 3.7 [40], butan-1-ol, D V1 = 3.7 [41], and propan-2-ol, D V1 = 2.2 around neutrality [13]. With ethanol, the e¡ect on D V1 /KA was smaller than on D V1 / KB , suggesting that NADþ binds before ethanol; the still signi¢cant size of D V1 /KA is probably due to dissociation of NADþ from the ternary complex [39]. With propan-2-ol and acetone, the kinetic mechanism is steady-state random in both directions [13]. A similar kinetic mechanism probably holds for most branched and secondary alcohols [34]. 5. Pre-steady-state kinetics Pre-steady-state kinetic studies provide the numerical values of the rate constants in the mechanism. The presteady-state kinetics of yeast alcohol dehydrogenase has been studied with the help of the KINSIM and FITSIM computer programs of Frieden [42^44]. These computer software packages can simulate the reaction progress curves and calculate the individual rate constants therefrom (Table 4). The magnitudes of the individual rate constants in Scheme 1 were calculated from reaction progress curves in both directions, keeping the concentration of reactants at such a level that dissociation of NADþ from the central complex was prevented, and therefore excluding the rate constants k11 ^k14 (Table 4) [45]. One can see from Table 4 that the magnitudes of rate constants obtained from the computer simulation of reaction progress curves [45] and from the steady-state kinetics [31] are very similar, the di¡erences re£ecting only the di¡erent enzyme preparations. In the horse liver enzyme, a large conformational change of the enzyme is triggered when the coenzyme binds, well documented both in structural terms [47] and by kinetic methods [48]. Recently, Northrop has reported that moderate pressure increases the capture of benzyl alcohol (V1 /KB ) in YADH-catalyzed oxidation of this alcohol with NADþ , by activating the hydride transfer step [49]. This means that the collision complex for hydride transfer (*EWNADþ ) has a smaller volume than the free alcohol plus the capturing form of the enzyme (EWNADþ ) [46]. This was a direct experimental proof for the isomerization step in the yeast enzyme, which enabled the estimation of the equilibrium constant k41 /k31 [75]; using this value, it was possible to calculate the equilibrium constant k42 /k32 (Table 4). Inspection of data in Table 4 clearly shows that, in the forward direction (oxidation of ethanol at neutral pH), the rate-limiting step is not the chemical reaction (k9 ), but the dissociation of NADH from the EQ-complex (k7 ). Like- FEMSYR 1513 11-11-02 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 wise, NADþ dissociates much faster from the EA-complex (k2 ) than NADH dissociates from the EQ-complex (k7 ). 6. Primary structure YADH-1 is a tetramer, composed of four identical subunits ; each subunit consists of a single polypeptide chain with 347 amino acids, with a molecular mass of 36 kDa [47]. Each subunit has one coenzyme-binding site and one ¢rmly bound zinc atom, which is essential for catalysis [50,51] ; the catalytic domain provides the ligands to this zinc atom: Cys-46, His-67 and Cys-174. The second zinc atom/subunit is liganded in a tetrahedral arrangement by four sulfur atoms from the cysteine residues 97, 100, 103 and 111; this zinc atom only has a structural role [52]. Table 5 shows the primary structures of the three isoenzymes of YADH [4,53^55]. The alignment of amino acid residues for all 47 members of the ADH family was made progressively rather than pairwise [1]. 7. The active site The amino acid sequences of horse liver alcohol dehy- Table 5 Primary structure of the three isoenzymes of yeast alcohol dehydrogenase 485 drogenase and YADH-1 are homologous, and the homology amounts to 25% of the amino acid residues [5]. YADH-1 has been crystallized, but only preliminary crystallographic studies have been reported [56]. The threedimensional structure of horse liver alcohol dehydrogenase in several binary and ternary complexes with coenzymes, substrates and inhibitors has been solved at high resolution [47]. The tertiary structures of liver and yeast enzyme are highly similar and able to accommodate extensive sequence changes between the enzymes [57]. Analogous to the liver enzyme, the subunits of the yeast enzyme are probably divided into two domains : the catalytic domain and the coenzyme-binding domain. The two domains are unequal in size; the catalytic domain contains 3/5 of all amino acids, whereas the coenzyme-binding domain contains the remaining 2/5 of the amino acids. The domains are separated by a cleft, containing a deep pocket which accommodates the substrate and the nicotinamide moiety of the coenzyme. One domain binds the coenzyme and the other provides ligands to the catalytic zinc, as well as to most of the groups that control substrate speci¢city [47]. Since the liver and yeast enzymes are homologous, molecular modeling of the yeast enzyme can approximate the structure of one subunit, but not yet the quaternary arrangement [57]. Fig. 1 shows a model of the active site of the yeast enzyme, drawn schematically after a model obtained in a molecular graphics display system by Plapp et al. [58]. The 3D-model of the active site of YADH provides an illustration of the main working machinery of the yeast enzyme. In order to perform catalysis, the active site of the enzyme has to bind a molecule of substrate and a molecule of coenzyme in a productive mode, and, subsequently catalyze a hydride-transfer reaction between them. The adenosine-binding site is easily accessible from solution, whereas the nicotinamide-binding site is situated at the center of the molecule, buried deep inside the protein [47]. Numerous amino acid residues in the primary structure of the enzyme are involved in substrate and coenzyme binding and in catalysis (Table 6). 7.1. Substrate-binding pocket The numbering of amino acids corresponds to horse liver alcohol dehydrogenase; alignment and numbering of amino acid in the yeast isoenzymes according to Sun and Plapp [1]. The inner wall of the pocket is lined with hydrophobic side chains from the residues Trp-57, Trp-93, Asn-110, Leu-132, Tyr-140, Thr-141, Met-294, Ala-296, and Ile318, which are from the same subunit as the zinc ligands. The substrate-binding site near zinc is narrow, because access is limited by Trp-93 and Thr-48. The voluminous amino acid side chains of Trp-57, Trp93 and Met-294 make the substrate-binding pocket in the yeast enzyme much more narrow than the corresponding pocket in the horse liver enzyme. FEMSYR 1513 11-11-02 486 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 7.2. Ligands to the active-site zinc At the bottom of the substrate-binding pocket, a zinc atom is coordinated to three protein ligands: two thiolates from Cys-46 and Cys-174, and one imidazole nitrogen of His-67. The other imidazole nitrogen of His-67 is hydrogen-bonded to a carboxylic group of Asp-49. A carboxylic group of Glu-68 is also in close proximity to the active-site zinc atom. Asp-49 and Glu-68 are the residues conserved in all known zinc-dependent alcohol dehydrogenases; instead of being inner-shpere ligands to the zinc, both amino acids are situated in the second sphere. The only polar groups in the pocket close to zinc are the zinc ligands, the nicotinamide moiety of the coenzyme, and the side chain of Thr-48. 7.3. Nicotinamide ring The nicotinamide ring binds in a cleft in the interior of the protein, close to the center of the molecule. On one side the ring interacts with Thr-178, Leu-203 and Met-294. The other side faces the active site, and is close to the catalytic zinc atom and the sulfur ligands of Cys-46 and Cys-174. The oxygen atom of the carboxamide group is hydrogen-bonded to the main-chain nitrogen atom of Val-319. The nitrogen atom of the carboxamide group is hydrogenbonded to the carboxyl oxygens of Val-292 and Ser-317. The side chain of Thr-178 helps to keep the nicotinamide ring of the nucleotide in the correct stereochemical position for hydride transfer (Fig. 5); Thr-178 is conserved in all known homologous alcohol dehydrogenases. Fig. 1. Model of the active site of yeast alcohol dehydrogenase, drawn schematically after Plapp et al. [58]. 7.4. The proton-relay system It was proposed by Eklund et al. [59] that the hydrogenbonded relay system in the liver enzyme (Fig. 3), Table 6 Positions of residues that participate in enzymatic functions of the yeast enzyme (adopted from Jo«rnval et al. [57]) Adenine binding pocket Interior Ser-198 Ile-250 Ile-222 Ser-269 Gly-224 Ala-274 Phe-243 Ala-277 Surface Ser-271 Ala-273 Adenosine^ribose binding Gly-199 Lys-228 Asp-223 Ser-269 Gly-225 Pyrophosphate binding His-47 Leu-203 Gly-202 Nicotinamide ribose Gly-293 Ser-269 Nicotinamide Thr-178 Substrate-binding pocket Trp-57 Thr-141 Trp-93 Met-294 Asn-110 Ala-296 Leu-132 Ile-318 Tyr-140 Proton relay system Thr-48 His-51 His 51::::NADþ ::::Ser 48::::CH 2 OHðH 2 OÞ::::Zn2þ ; stretching from His-51 on the surface of the enzyme to the active-site zinc atom in the interior of the enzyme, serves as a proton conductor which helps the dissociation of alcohol to alcoholate in the productive ternary enzymeWNADþ Walcohol-complex. The yeast enzyme has the same proton relay system with, however, Ser substituted with Thr (Fig. 1) [58]. Therefore, since the enzymeWNADþ Walcoholate-complex is considered a true transition state in the yeast enzyme catalysis [60], the proton relay system must greatly accelerate the same. 7.5. Binding of the coenzyme Ligands to active-site zinc atom Inner sphere Cys-46 Cys-174 His-67 Second sphere Asp-49 Glu-68 The coenzyme is bound to the apoenzyme by numerous secondary valence forces. Important amino acid residues are: Asp-223, which is hydrogen-bounded to AMP-ribose, His-47, forming a salt bridge with AMP-orthophosphate, and Leu-203, forming a hydrogen bond to NMN-orthophosphate. FEMSYR 1513 11-11-02 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 487 8. Mutations in the yeast enzyme 8.2. Ligands to the active-site zinc (Asp-49, Glu-68) The three yeast ADH genes have been cloned and described [4,54,55]. Therefore, it was possible to change individual amino acids in the primary structure of YADH-1 via site-directed mutagenesis and isolate a quantity of mutated enzymes (Table 7). In recent years, a number of genetically engineered mutants of YADH-1 were isolated and kinetically characterized, principally by Plapp and his co-workers. Most of these mutations involve amino acids which are intimately involved in the binding of substrates and in catalysis, and provide information about the general principles concerning the function of the catalytic residues. Table 7 shows the steady-state kinetic properties of all YADH mutants described so far participating in substrate binding and in catalysis. The carboxylate group of Asp-49 is hydrogen-bonded to His-67, which in turn coordinates the active-site zinc ; in addition, the carboxylate group of Glu-68 is in the vicinity of the active-site zinc. If Asn is substituted for Asp-49 or Gln for Glu-68, a negative charge is removed from the vicinity of the active-site zinc; these substitutions reduce the catalytic e⁄ciency with ethanol (V1 /KB ) 1000 times and 100 times, respectively, and the catalytic constant (V1 ) 40 times. These reductions in activity were interpreted by an increased electrostatic potential near the active-site zinc, due to removal of negative charges; as a consequence the activity is decreased by hindering isomerizations of enzyme^substrate complexes [39]. 8.1. Substrate-binding pocket (Met-294, Trp-57, Trp-93) An exchange of Ser for Thr-48 does not interrupt the hydrogen bonding in the proton relay system and, as expected, the activity of the Thr48Ser mutant is very similar to that of the wild-type. The double mutant Thr48Ser: Trp93Ala and the triple mutant Thr48Ser:Trp57Met : Trp93Ala show decreased activities that are obviously due to removal of bulky tryptophan residues from the substrate-binding pocket [20]. An exchange of Cys or Ala for Thr-48 disrupts the hydrogen bonding in the relay system and, as expected, renders the enzyme inactive [58]. The role of His-51 in catalysis has been tested by replacing it with glutamine or glutamic acid [58,61]. These residues have an appropriate size to form the hydrogen bond with the 2P-hydroxyl group of the nicotinamide ribose; thus, binding of the coenzyme in the mutant enzymes could resemble binding in the wild-type enzyme. On the other hand, a glutamine residue would not be able to An exchange of Leu for Met-294, on the edge of the substrate-binding pocket, has very little in£uence on the steady-state kinetic properties of the enzyme with ethanol or acetaldehyde. On the other hand, the Met294Leu mutant has a 10-fold lower catalytic activity (V1 ) with butan1-ol, indicating that the C4-atom of butan-1-ol is in a close contact with Met-294, whereas the shorter ethanol is not [9]. An exchange of Met or Leu for Trp-57 decreases the catalytic e⁄ciency (V1 /KB ) with ethanol only three- to four-fold, whereas an exchange of Ala for Trp-93 decreases the catalytic e⁄ciency 300-fold; with an enlargement of the substrate-binding pocket in the latter case, the enzyme acquires weak activity with branched-chain alcohols (2-methyl-1-butanol, 3-methyl-1-butanol) and benzyl alcohol [19,20]. 8.3. The proton-relay system (Thr-48, His-51) Table 7 Steady-state kinetic constants for YADH mutants, with ethanol and acetaldehyde as substrates, determined at pH 7.3, 30‡C Mutant V1 (s31 ) V1 /KA (mM31 s31 ) V1 /KB (mM31 s31 ) V2 (s31 ) V2 /KQ (mM31 s31 ) V2 /KP (mM31 s31 ) Source YADH-1 Substrate-binding pocket Met294Leu Trp57Met Trp57Leua Trp93Ala Ligands to the active-site zinc Asp49Asn Glu68Gln The proton relay system Thr48Ser Thr48Ser :Trp93Ala Thr48Ser :Trp57Met:Trp93Ala Thr48Cys Thr48Ala His51Gln His51Glu 340 2000 20 1700 15 500 1545 [9] 500 220 99 110 794 265 91 48 26.3 4.9 7.4 0.07 2100 1900 211 ND 26 250 6 790 2 245 ND 2100 513 ND ND [9] [20] [19] [20] 7.5 9.9 0.83 24 0.02 0.24 113 730 125 4 560 2.3 13 [39] [39] 200 140 120 61 61 27 2 2200 11.8 152 0.033 22.2 0.75 No detectable activity No detectable activity 245 1.4 26 0.26 1500 530 ND 13 640 4 077 ND 2027 5.7 ND 2800 ND 25 450 ND 215 ND [20] [20] [20] [58] [58] [61] [58] ND = not determined. a Determined at pH 8.2, 25‡C. FEMSYR 1513 11-11-02 488 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 participate in base catalysis, whereas a glutamate residue could accept a proton. Plapp et al. [58] have found that a wild-type enzyme has a distinct pKa value of 7.7 in the pH-pro¢le for the V1 /KB function. Replacement of His-51 with Gln or Glu reduces the value of V1 /KB 13-fold and 60-fold at pH 7.3, respectively; in addition, the pKa value of 7.7 in the pH pro¢le of the V1 /KB function is abolished in both cases. These results were interpreted by a mechanism in which the amino acid residue in the mutant enzyme hinders the deprotonation of alcohol through the proton relay system [58]. On that interpretation, these results are consistent with the role of His-51 in the proton relay system, where it participates as a base. to the free enzyme (k8 ) decreases in alkaline over a single pKa value 7.8, while the dissociation rate constant for the EWNADH-complex (k7 ) is almost pH-independent, from pH 6.5 to 9.0. In recent years, a number of genetically engineered mutants of YADH-I, with mutations in the adenylate-binding pocket, have been isolated and kinetically characterized, principally by Plapp and his co-workers (Table 8). The following general conclusions may be drawn from the kinetic data shown in Table 8. 9.1. Adenine site substitutions (Ser-198, Gly-224, Gly-225) Gly224Ile and Gly225Arg mutants have only modest e¡ects on coenzyme binding and other kinetic constants, but the Ser198Phe mutant signi¢cantly decreases its a⁄nity for coenzymes and turnover numbers. 9. Binding of coenzymes Fig. 2 summarizes the steady-state and ligand-binding data relevant for the binding of coenzymes to the free enzyme. The dissociation constant of the EWNADþ complex for the yeast enzyme, Kþ E;NAD , is practically pH-independent; on the other hand, the dissociation constant of the EWNADH complex, KE;NADH , decreases with lower pHvalue over three apparent pKa values (6.6, 8.0 and 9.0). The association rate constant for the binding of NADH 9.2. Adenosine^ribose binding (Asp-223) The Asp223Gly:Gly225Arg double mutant shows a decrease in all kinetic parameters, but uses NAD(H) and NADP(H) with about the same e⁄ciency. 9.3. Pyrophosphate binding (His-47, Ala-200, Leu-203, Gly-204) Mutation of the residues Ala-200, Leu-203 or Gly-204 decreases all kinetic parameters signi¢cantly, suggesting that these amino acids are essential for the binding of the pyrophosphate moiety of the coenzyme. On the other hand, substitution of His-47 by the basic amino acid Arg decreases the catalytic activity with NAD(H) only modestly. 9.4. Nicotinamide^ribose binding (Ser-269) The Ser269Ile mutant decreases its turnover numbers by 350-fold. Studies of the mutants in the adenylate-binding site of the enzyme show that several amino acid residues at the proposed adenylate-binding site of the enzyme are important for coenzyme binding and formation of productive ternary complexes. The Asp223Gly:Gly225Arg double mutant was the only mutant that uses NAD(H) and NADP(H) with about the same e⁄ciency ; this result suggests that conversion of the coenzyme speci¢cally requires multiple substitutions [62]. Mutations of amino acids Leu203 and Thr-178 have been performed in order to locate the structural determinants of the high stereospeci¢city of the enzyme for the coenzyme NAD(H) [64]. Fig. 2. pH pro¢les for the binding parameters of coenzymes to the free enzyme; rate constants k7 and k8 , as in Scheme 1 (adopted from Leskovac et al. [34]). 10. Chemical mechanism Primary structure, tertiary structure and point muta- FEMSYR 1513 11-11-02 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 489 Table 8 Steady-state kinetic constants for YADH mutants in the adenylate binding pocket, with ethanol and acetaldehyde as substrates, determined at pH 7.3, 30‡C Mutant V1 (s31 ) V1 /KA (mM31 s31 ) V1 /KB (mM31 s31 V2 (s31 ) V2 /KQ (mM31 s31 ) V2 /KP (mM31 s31 ) Source YADH-1 Adenine site substitutions Ser198Phe Gly224Ile Gly225Arg Adenosine^ribose binding Asp223Gly Asp223Gly:Gly225Arg Pyrophosphate binding Leu203Alaa Leu203Ala :Thr178Sera His47Arg Gly204Ala Ala200v:Ala201Leub NMN^ribose binding Ser269Ile 340 2000 20 1700 15 500 1 545 [9] 40 360 550 1.25 414 1222 0.14 16 21 150 4000 2400 25 6 060 12 000 71 20 000 18 000 [62] [62] [62] 38 17 2.1 0.94 0.2 0.13 300 110 60 18.33 75 20 [63] [63] 106 31.9 60 8 67 56.4 61.3 400 0.26 13.13 ND ND 0.9 0.02 1.4 ND ND 460 200 ND ND ND 46 000 25 ND ND ND 98 11 ND [64] [64] [32] [62] [62] 1.0 0.36 0.003 5.4 13.85 0.31 [62] ND = not determined. a Determined at pH 8.2, 25‡C. b Alignment of amino acids according to Table 5. Ala200 is an insertion in the yeast enzyme with respect to other members of the alcohol dehydrogenase family of enzymes; therefore, this residue is not counted in the primary structure that follows after this residue [1]. tions in the yeast enzyme, outlined in the preceding sections, strongly suggest that the integrity of the proton relay system is indispensable for the activity of the enzyme. Based on this integrity of the relay system, which is maintained throughout the catalytic cycle, Cook and Cleland [60] have proposed the chemical mechanism of action for the yeast enzyme as shown in Scheme 2. In this mechanism, B and P represent alcohol and ketone, and k3 , k4 , k5 and k6 represent hydride-transfer steps ; X is an intermediate with the stoichiometry of an alkoxide, and k1 and k2 are the steps in which a proton is transferred from B to a group on the enzyme to give X, and similarly for the reverse process. An assignment of appropriate pKa values to all dissociation forms of the enzyme in Scheme 2 was founded on studies of the pH dependence of the steady-state kinetics and ligand-binding parameters [14,16,26,33^36,65], as outlined below. Table 9 shows the macroscopic pKa values calculated from the pH pro¢les of the maximal rates (V1 ) and the speci¢city constants (V/K) with various substrates. Table 10 presents the pKa values calculated from the pH pro¢les of binding constants (Ki ) for competitive dead-end inhibitors. Scheme 2. The speci¢city constants V/K with ‘nonsticky’ substrates, such as propan-1-ol, propan-2-ol, NDMA, DACA and acetone, provide information on catalytically active groups in enzyme^coenzyme complexes [66], if the pH pro¢les of V/K are ¢tted to initial-rate equations appropriate to the mechanism in Scheme 2 [36]. In this way, the pK1 (8.0) and pK5 (7.9^8.0) values in Scheme 2 were estimated. From the binding of azide, a dead-end inhibitor competitive with alcohols, the value for pK1 (7.9) was con¢rmed; from the binding of acetamide, a dead-end inhibitor competitive with aldehydes, the values for pK4 (8.3) and pK5 (7.9) were estimated. pH pro¢les for the V1 function provide information on catalytically active groups in the productive ternary enzymeWNADþ Walcohol-complex [66]. In this way the pK2 value was estimated (8.3), from the pH pro¢les of V1 with butan-1-ol and propan-2-ol. An indirect estimation provided the value of pK3 (8.3) [36]. The chemical mechanism of action, presented in Scheme 1, can be drawn entirely in terms of the proton relay system, as is shown in Fig. 3; in Fig. 3, however, the Thr-48 residue was omitted from the relay for the sake of simplicity. The key feature of Fig. 3 is that His-51 lies at the surface of the protein and thus can be deprotonated as in the conversion of HEAX to EAX or HEQP to EPQ, while reactants are bound and the state of protonation of molecules in the substrate-binding site is locked. Thus, HEAX can be deprotonated to EAX without preventing subsequent hydride transfer. A di¡erent view on the chemical mechanism of action of yeast alcohol dehydrogenase has been presented by Bra«nde¤n et al. [5]. These authors proposed that the Zn2þ -bound FEMSYR 1513 11-11-02 490 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 Table 9 Macroscopic pKa values and pH-independent limiting constants in various YADH-catalyzed reactions (adopted from Leskovac et al. [36]) Substrate pKa Butan-1-ol V1 (s31 ) 6.1 7.3 8.3 Propan-2-ol 6.2 V1 /KB (s31 ) 7.4 8.3 Propan-1-ol 6.7 V1 /KB (mM31 s31 ) 7.4 8.2 Propan-2-ol 6.5 V1 /KB (M31 s31 ) 7.1 7.8 Acetone 7.9 V1 /KB (M31 s31 ) 8.2 9.0 DACA 8.0 V2 /KP (mM31 s31 ) 8.0 NDMAa V2 /KP (mM31 s31 ) Limiting constant Dixon^Webb plot 191 increases with pH 81 increases with pH 9.0 increases with pH 155 increases with pH 6.9 decreases with pH 0.25 decreases with pH 2.2 0.9 plateau at low pH plateau at high pH water dissociates when the coenzyme NADþ is added; the remaining (OH)3 deprotonates the alcohol, which is then bound to the Zn2þ ion as the fourth ligand. Fig. 4 shows this dissociation in the proton relay system. Recently, Nadolny and Zundel [67] have claimed experimental evidence supporting the above mechanism. These authors obtained Fourier-transform infrared (FTIR) spectra of various complexes of yeast alcohol dehydrogenase with NADþ and coenzyme analogs ; from the FTIR spectra they concluded that, upon binding of NADþ to the enzyme, N1 of the coenzyme adenosine becomes protonated and the molecule of water in the active site dissociates to a hydroxyl anion. It was postulated that the positive charge is conducted from the zinc-bound water to histidine-51 and then further to the N1-atom of the adenine rest via the proton relay system through the protein. Thus the binding of NADþ to the enzyme shifts the equilibrium 1C2 in Fig. 4 to the right. The substrate, alcohol, is then deprotonated by the (OH)3 bound to the Zn2þ ion and forms the structure 3. The experiments of Nadolny and Zundel [67] with the yeast enzyme were conducted at pH 7.5 and they do not explain the pH dependence of the enzyme activity. Further, the proposed mechanism lacks the explanation for the conductance of the positive charge from His-51 to \. adenine across a distance of approximately 7 A hydrogen (pro-R or A-type) at the 4-position of NADH to the carbonyl carbon of the substrate (Fig. 5). The stereochemical ¢delity of the hydride transfer reaction is very high, and YADH makes but one stereochemical ‘mistake’ every 7 000 000 turnovers. If the bulky side chain of Leu-203 is exchanged with Ala, the Leu203Ala mutant (Table 7) makes one stereochemical ‘mistake’ every 850 000 turnovers with NADH, and every 450 turnovers with thio-NADH, which has a weaker hydrogenbonding capacity. From this, it was concluded that the decrease in stereochemical ¢delity comes from an increase in the transfer rate of the 4-Si-hydrogen of NADH. The nicotinamide ring of the coenzyme is kept in a correct position for hydride transfer mainly by hydrogen bonds between its amide group and Val-292 and Val-319, and the rotation of 180‡ around the glycosidic bond is obstructed mainly by the side chain of Leu-203 [64]. The main reaction catalyzed by alcohol dehydrogenase is, in principle, a very simple reaction. An alcohol group is oxidized by the removal of a proton from the hydroxyl group and by the transfer of a hydride ion from the adjacent carbon atom to NADþ . By analogy with the horse liver enzyme [47], we may assume that hydride transfer in the yeast enzyme occurs in a completely water-free environment. Direct transfer of a hydride ion is facilitated in a hydrophobic environment, where water is excluded. The positive charge on the nicotinamide ring is crucial for the enhanced binding of alcohol to the enzyme ; insertion of the positive charge in this hydrophobic environment facilitates formation of the negatively charged alcoholate ion. The creation of an alcoholate ion greatly facilitates hydride transfer. The important role of the zinc atom in alcohol oxidation is to stabilize the alcoholate ion for the hydride-transfer step. In the reverse direction, zinc functions as an electron attractor, which gives rise to an increased electrophilic character of the aldehyde, consequently facilitating the transfer of a hydride ion to the aldehyde. Thus, the proposed mechanism is essentially electrophilic catalysis mediated by the active-site zinc atom. The overall oxidation of alcohol to aldehyde involves the net release of one proton (Eq. 1); the ultimate source of this proton is alcohol. The release of a proton from the bound alcohol occurs in the center of the enzyme molecule in a region that is inaccessible to solution ; the proton is transferred by certain groups on the enzyme to the surrounding solution (Fig. 1). Because water is not directly Table 10 Macroscopic pKa values and pH-independent constants for ternary complexes of YADH with competitive dead-end inhibitorsa 11. Hydride transfer Complex One of the classical aspects of coenzyme binding to yeast alcohol dehydrogenase is the A-stereospeci¢city of the coenzyme [68]. YADH-catalyzed reactions are highly stereospeci¢c ; the enzyme catalyzes the transfer of the Re- þ þ EWNAD +Az1EWNAD WAz EWNADH+AA1EWNADHWAA a pKa Limiting constant 7.9 8.3 0.95 mM (at low pH) 45.8 mM (low pH) 118 mM (high pH) Calculated from the data of Leskovac et al. [35]. FEMSYR 1513 11-11-02 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 491 Fig. 3. The chemical mechanism of action of alcohol dehydrogenase [36]. involved in the catalytic reaction, that is, no hydrolysis or hydration, there is no reason to suggest a role for a water molecule at the active site of YADH [36]. In catalysis, the molecules of the substrate and the nicotinamide ring of the coenzyme probably do not have ¢xed positions. The rearrangement of electron con¢guration on the carbon atom from the sp2 hybridization in aldehyde to the sp3 in alcohol, requires di¡erent pathways for hydride transfer and, consequently, di¡erent relative orientations [69]. Primary and secondary kinetic isotope e¡ects (kH /kD , kH /kT and kD /kT ) in YADH-catalyzed reactions have been studied as a probe of quantum-mechanical hydrogen tunneling. Hydrogen tunneling was ¢rst suggested in YADH-catalyzed reactions following the measurement of anomalously large secondary kinetic isotope e¡ects [70]. In the absence of tunneling and coupled motion, a secondary kinetic isotope e¡ect is expected to be intermediate between unity and the value of the equilibrium isotope e¡ect [71]. Thus, Cook et al. [70] studied the oxidation of 2propanol with [4-2 H]NADþ , ½4 2 HNADþ þ CH3 CHðOHÞCH3 ! ½4S 2 HNADH þ CH3 COCH3 þ Hþ ð7Þ and found a secondary kinetic isotope e¡ect of 1.22 and an equilibrium isotope e¡ect of 0.89, which is indicative of hydrogen tunneling. In hydride-transfer reactions catalyzed by YADH, one can measure the kH /kD , kH /kT , and kD /kT primary, and, similarly, the secondary isotope e¡ects. The Swain^Schaad relationship states that, in the absence of tunneling and coupled motion, and without kinetic complexities, ðkD =kT Þ3:30 ¼ kH =kT ð8Þ Klinman [72] has argued that, out of various thermody- namic and kinetic criteria, the most sensitive one for detection of tunneling is the breakdown of the above relationship, particularly for the secondary kinetic isotope e¡ect [73]. In line with this, Cha et al. [74] have studied the oxidation of benzyl alcohol by NADþ , using six di¡erently labeled alcohols as substrates : ½14 CC 6 H 5 CH 2 OH; ½14 CC 6 H 5 CD2 OH; C 6 H 5 CðH; TÞOH; C 6 H 5 CðT; HÞOH; C 6 H 5 CðD; TÞOH; C 6 H 5 CðT; DÞOH in order to obtain all combinations of isotope e¡ects. This reaction is suitable for exploring hydrogen tunneling because of the lack of any kinetic complexity, as it has a rate-limiting H-transfer step [75]. Cha et al. [74] have demonstrated that, in this reaction, the exponents in Eq. 8 are 3.58 and 10.2 for the primary and secondary kinetic isotope e¡ect, respectively, indicating signi¢cant breakdown of the semi-classical upper limit. For hydrogen tunneling to occur, the reactive carbon atoms have been brought close together so that the classical energy barrier is penetrated. Thus, it appears that hydrogen tunneling is an additional general phenomenon which facilitates the YADH catalysis [23,76^78]. Leskovac et al. [79] have studied the primary kinetic isotope e¡ects and the internal thermodynamics of the YADH-catalyzed oxidation of 2-propanol-h8 and 2-propanol-d8 with NADþ ; the properties of this reaction were compared with non-enzymatic model redox reactions of N1 -substituted-1,4(1 H2 )dihydronicotinamides and N1 -substituted-1,4(1 H2 H)dihydronicotinamides with a number of various oxidizing agents. The kinetic and thermodynamic properties of the enzymatic reaction closely resemble the model hydride-transfer reactions which probably proceed via a linear transition state, and are very dissimilar from Fig. 4. The Bra«nde¤n mechanism. FEMSYR 1513 11-11-02 492 V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494 [8] [9] [10] [11] [12] [13] Fig. 5. Stereospeci¢city of YADH catalysis. NADH binds anti, presenting Re-hydrogen (HRe ) to acetaldehyde lying above the coenzyme in this diagram. For clarity, Thr-178 is not shown; the methyl group of this side chain lies below and to the left of the nicotinamide behind Leu-203 (reproduced from Weinhold et al. [64], with permission of the corresponding author). [14] [15] [16] reactions which proceed via a bent transition state, suggesting that this particular enzymatic reaction has a linear transition state. [17] [18] Acknowledgements [19] This work was ¢nancially supported by the Ministry of Science and Technology of the Republic of Serbia. [20] References [21] [1] Sun, H.-W. and Plapp, B.V. (1992) Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. J. Mol. Evol. 34, 522^535. [2] Young, E.T., Williamson, V., Taguchi, A., Smith, M., Sledziewski, A., Russel, D., Ostermann, J., Denis, C., Cox, D. and Beier, D. (1982) The alcohol dehydrogenase genes from the yeast, Saccharomyces cerevisiae : isolation, structure and regulation. 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