Kinetic mechanism of penicillin V acylase activation byshort-chain alcohols

Alcohol-induced activation of penicillin V acylase from Streptomyces lavendulae by some water-miscible primary alcohols such as methanol, ethanol, 1-propanol and 1-butanol has been extensively studied. The effects of various concentrations of these alcohols on the maximum velocity of penicillin V hydrolysis (Vmax) and the apparent Michaelis-Menten constant (Km) of the enzyme are reported. Lineweaver-Burk analyses of the kinetic data revealed a phenomenon of nonessential activation in the presence of short-chain primary alcohols. In the concentration range where activation was observed, conventional secondary replots of 1/Δ slope or 1/Δ intercept versus 1/[alcohol] yielded perfect straight lines, demonstrating the binding of a single molecule of alcohol to an activator site in penicillin V acylase. Furthermore, the enzyme displayed a propensity to bind the longer chain alcohols, as reflected by the dissociation constant KA values of 493, 369, 228 and 11.8 mM for methanol, ethanol, 1-propanol and 1-butanol, respectively. Therefore, 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 another one, which is catalytically more effective than the alcohol-free form of the penicillin acylase. Further addition of alcohol results in a competitive inhibition of the enzyme in its activated state. An inverse relationship between the chain length of the alcohols and the breakpoints in the biphasic normalized vmax/Km ratio versus alcohol concentration plots was observed.