Substituent Effects on the Pre‐Steady‐State Kinetics of Oxidation of Benzyl Alcohols by Liver Alcohol Dehydrogenase

Abstract

1. The oxidation of benzyl alcohols by liver alcohol dehydrogenase under conditions [S], $-[El, is a biphasic process with exponential rise to a steady state. The rate constants for the transient phase were determined by a curve-fitting procedure.2. A large isotope effect was obtained for p-methoxybenzyl alcohol, indicating that hydrogen transfer is rate-determining for the transient phase. Rate constants for the hydrogen transfer step, k3, were obtained for the various alcohols and correlated with the Hammett o constant by plotting log k3 against o. The slope of this line (e = -0.76) is consistent with rate-limiting hydride transfer; a mechanism is proposed to account for the low magnitude of e. 3.The pre-steady-state rate constants, kb, for all benzyl alcohols (c 20 s-') are lower than that for ethanol. 2-Methylpropan-1-01 and cyclohexylmethanol have pre-steady-state rate constants of about 150 s-', indicating that the lower kh values for benzyl alcohols are due to the electronwithdrawing effect of the benzene ring, rather than to steric effects.Steady-state and transient kinetic studies on liver alcohol dehydrogenase have established that the reaction proceeds by an ordered mechanism with coenzyme binding first, and coenzyme dissocation as the rate-determining step at saturating concentrations [I -41. The existence of ternary complexes in the reaction mechanism was indicated by product inhibition studies [4,5]. Subsequently Shore and Gutfreund [6] studied the pre-steady-state formation of NADH during the oxidation of ethanol by alcohol dehydrogenase in order to measure the rate of interconversion of the ternary complexes. They found a substantial kinetic isotope effect on the rate constant for initial formation of enzyme-bound NADH, indicating that the hydrogen transfer step was ratelimiting for the transient phase [6].Substrate structure has been found to have a marked effect on the rate of the hydrogen transfer step [7]. A pre-steady-state burst of bound NADH was observed [7] during the oxidation of two primary alcohols, ethanol and I-propanol ; however no burst was observed for methanol or the secondary alcohol, 2-propanol, because for these alcohols the hydrogen transfer step was slower than NADH dissociation and hence rate-determining for the steady-state phase.~.

pH, isotope, and substituent effects on the interconversion of aromatic substrates catalyzed by hydroxybutyrimidylated liver alcohol dehydrogenase

“…Interpretation of Substituent Effects. Hardman et al (1974) and we have found small, negative p values (p+ = -0.2 to -0.4) for the oxidation of substituted benzyl alcohols. Furthermore, Blackwell and Hardman (1975) found a value of p* of -1.8 for a series of different primary alcohols.…”

“…The magnitudes of the isotope effects indicate that transfer of hydrogen in the ternary complexes is at least partially rate limiting in the steady-state turnover. Although the isotope effects are not large, they are the same as those obtained from transient kinetic studies with the liver enzyme (Hardman et al, 1974;McFarland and Bernhard, 1972;Jacobs et al, 1974) and almost as large as those obtained from steady-state studies with the yeast enzyme (Klinman, 1972(Klinman, , 1975. Given that transfer of hydrogen is at least partially rate limiting, the mechanism of hydroxybutyrimidylated enzyme acting on benzyl alcohol may not be a compulsory order, but rather a preferred order, mechanism, as was found previously for the native enzyme acting on secondary alcohols (Dalziel and Dickinson, 1966;Dalziel, 1975).…”

“…It has been suggested that the small p values observed for the alcohol dehydrogenases are consistent with mechanisms in which the transfer of a hydride ion from one substrate to the other is coupled with the transfer of a proton mediated by a group on the enzyme acting as an acid-base catalyst (Hardman et al, 1974;Blackwell and Hardman, 1975;Klinman, 1975Klinman, , 1976. Such a mechanism is discussed in detail below.…”

“…Jacobs et al (1974) found a small positive p value for reduction of some benzaldehydes, which led them to suggest that there was a highly positively charged transition state; from this result, a large negative p value for the oxidation of the benzyl alcohols is predicted. But Hardman et al (1974) found a small negative p value for the alcohols and suggested that a hydride ion and a proton are transferred synchronously.…”

Transition-state structure in the yeast alcohol dehydrogenase reaction: the magnitude of solvent and .alpha.-secondary hydrogen isotope effects

“…Funderburk & Jencks (1978) have recently discussed the imbalance in values of Bronsted a's and /3's, p values, and «-secondary isotope effects for the acid-catalyzed breakdown of carbinolamines to semicarbazones; as pointed out by these authors, secondary isotope effects indicate little or no bond rehybridization toward the sp2 carbon atom in the transition state, in contrast to p values, which support a large degree of carbon-oxygen bond cleavage in the transition state. An examination of the correlation between electronic substituent effects and secondary isotope effects for the addition of nucleophiles to aldehydes under a variety of experimental conditions would provide important reference information toward the interpretation of the data in the yeast alcohol dehydrogenase reaction.3 A comparison of structure-reactivity correlations in the horse liver alcohol dehydrogenase to the yeast enzyme indicates a similar (possibly identical) distribution of charge at C-l of substrate in the transition state (Hardman et al, 1974). Although «-secondary isotope effects have not yet been reported for the horse liver system, measurement of these effects would provide a subtle test of the extent to which transitionstate structure has been conserved among enzymes from divergent evolutionary sources catalyzing the same reaction.…”