Activity of yeast alcohol dehydrogenases on benzyl alcohols and benzaldehydes

Abstract

The substrate specificities of yeast alcohol dehydrogenases I and II from Saccharomyces cerevisiae (SceADH1 and SceADH2) and Saccharomyces carlsbergensis (ScbADH1) were studied. For this work, the gene for the S. carlsbergensis ADH1 was cloned, sequenced and expressed. The amino acid sequence of ScbADH1 differs at four positions as compared to SceADH1, including substitutions of two glutamine residues with glutamic acid residues, and has the same sequence as the commercial yeast enzyme, which apparently is prepared from S. carlsbergensis. The electrophoretic mobilities of ScbADH1, SceADH2 and commercial ADH are similar. The kinetics and specificities of ScbADH1 and SceADH1 acting on branched, long-chain and benzyl alcohols are very similar, but the catalytic efficiency of SceADH2 is about 10 to 100-fold higher on these substrates. A three dimensional structure of SceADH1 shows that the substrate binding pocket has Met-270, whereas SceADH2 has Leu-270, which allows larger substrates to bind. The reduction of a series of p-substituted benzaldehydes catalyzed by SceADH2 is significantly enhanced by electronwithdrawing groups, whereas the oxidation of p-substituted aromatic alcohols may be only slightly affected by the substituents. The substituent effects on catalysis generally reflect the effects on the equilibrium constant for the reaction, where electron-withdrawing substituents favor alcohol. The results are consistent with a transition state that is electronically similar to the alcohol, supporting previous results obtained with commercial yeast ADH.

Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol

“…Similar changes in mobility were observed for the D49N and E67Q enzymes [9]. For reference, commercial yeast ADH, which has two additional negatively-charged residues, migrates more rapidly [18]. …”

“…The high pH is required to separate the wild-type enzyme (with histidine residues) from the forms that retain an additional positive charge above pH 9 (as indicated on the figure). Commercial yeast ADH, which comes from Saccharomyces carlsbergensis , has 2 additional negative charges [18]. The E67Q enzyme also migrates with more positive charge [9].…”

The use of kinetic isotope effects in studies of hydrogen transfers

“…An early attempt to measure this KIE produced results within error of unity, which did not surprise the authors because the KIE in the forward direction (alcohol to aldehyde) was equal to the EIE, suggesting a TRS close to the aldehyde. 59 Our measurements differ from unity and appear to be quite precise as a result of using only the aromatic-active isozyme of yADH, 123 and a broad range of time points.…”

“…returned what appear to be contradictory results, some suggesting an early TS, while others point to a late TS. 57,58,122,123 In pioneering studies, Klinman measured linear free energy relationships (LFERs) using benzyl substrates in both forward and reverse directions, and found evidence to support an alcohol-like TS, 57,58 and 2° hydrogens (Figure 3.1). 26 Subsequently, H tunneling appeared to be a common feature for H transfer in both enzymatic and nonenzymatic reactions.…”

“…While most previous studies 31,[57][58][59]141 used commercially available yADH, a recent report found that commercially available materials are mixtures of isozymes, only one of which is active toward aromatic substrates. 123 The aromaticactive isozyme, yADH 2, was expressed and purified as described previously 142 benzaldehyde (300,000 dpm 14 C and 1,500,000 dpm 3 H), 80 mM glycine (pH 8.5), 20 mM NADH (or NADD), and sufficient enzyme to reach complete conversion to products within a few hours. Reactions were initiated by addition of the enzyme and maintained at 25° until quenching with 10 mM HgCl 2 at a range of time points.…”