Kinetic mechanism of penicillin V acylase activation by short-chain alcohols

Alcohol-induced activation of penicillin V acylase from Streptomyces lavend ulae by some water-miscible primary alcohols such as methanol, ethanol, I-p ropanol and 1-butanol has been extensively studied. The effects of various concentrations of these alcohols on the maximum velocity of penicillin V hy drolysis (V-max) and the apparent Michaelis-Menten constant (K-m) of the en zyme are reported. Lineweaver-Burk analyses of the kinetic data revealed a phenomenon of nonessential activation in the presence of short-chain primar y alcohols. In the concentration range where activation was observed, conve ntional secondary replots of 1/Delta slope or 1/Delta intercept versus 1/[a lcohol] yielded perfect straight lines, demonstrating the binding of a sing le molecule of alcohol to an activator site in penicillin V acylase. Furthe rmore, the enzyme displayed a propensity to bind the longer chain alcohols, as reflected by the dissociation constant K-A. values of 493, 369, 228 and 11.8 mM for methanol, ethanol, I-propanol and 1-butanol, respectively. The refore, we have proposed a high affinity activator site for alcohols in the enzyme. Very hydrophobic alcohols such as 1-butanol bind more efficiently to this activator site and convert the enzyme form one conformation to anot her one, which is catalytically more effective than the alcohol-free form o f the penicillin acylase. Further addition of alcohol results in a competit ive inhibition of the enzyme in its activated state. An inverse relationshi p between the chain length of the alcohols and the breakpoints in the bipha sic normalized V-max/K-m ratio versus alcohol concentration plots was obser ved. (C) 2001 Elsevier Science Inc. All rights reserved.