Phenyl ester hydrolysis catalysed by alcalase. Case for electrophilic participation at ester carbonyl oxygen

Abstract

Kinetic parameters for the hydrolysis of substituted phenyl acetates and hippurates catalysed by alcalase have been measured at 25° and 0·1M ionic strength. The pH-dependencies of the k₀/K_m parameter for 4-methoxy- and 4-chloro-phenyl hippurates and 4-nitrophenyl acetate are identical and sigmoid in shape for the three substrates. Acylation is probably the rate-limiting step in methyl hippurate hydrolyses because the value of k₀ is less than the essentially constant value for the aryl and benzyl esters. Acylation (k₀/K_m is used as a measure) is insensitive to the σ value for aryl hippurates (ρ= 0·35, r= 0·788) but more sensitive for the acetates (ρ= 1·14, r= 0·868). Electrophilic assistance by the enzyme at the ester carbonyl oxygen atom is advanced to explain part of the catalytic activity. Binding of the leaving group (aryl or benzyl) to the enzyme by lipophilic forces is also suggested to account for part of the efficiency.