ACTIVE-SITE OF BEE VENOM PHOSPHOLIPASE A(2) - THE ROLE OF HISTIDINE-34, ASPARTATE-64 AND TYROSINE-87

In bee venom phospholipase A(2), histidine-34 probably functions as a Bronsted base to deprotonate the attacking water. Aspartate-64 and tyr osine-87 form a hydrogen bonding network with histidine-34. We have pr epared mutants at these positions and studied their kinetic properties . The mutant in which histidine-34 is changed to glutamine is catalyti cally inactive, while the mutants in which aspartate-64 is changed to asparagine or alanine (interfacial turnover numbers are reduced by 50- 100-fold) or in which tyrosine-87 is changed to phenylalanine (no chan ge in turnover number) retain good activity. The interfacial Michaelis constants are changed by less than 10-fold for all mutants. Molecular simulations suggest that mutation of aspartate-64 and tyrosine-87 sho uld yield enzymes that retain a native-like structure and support cata lysis. The pK(a) of the histidine-34 imidazole was deduced from the pH -rate profile and from the pH dependence of the rate of histidine-34 a lkylation by 2-bromo-4'-nitroacetophenone. The pK(a) is increased abou t one-half unit by the tyrosine-87 mutation and reduced about one-half unit by the aspartate-64 to asparagine mutation, while in the asparta te-64 to alanine mutant the pK(a) is unchanged. These pK(a)s are gener ally consistent with results of electrostatic calculations and suggest that the hydrogen bond between aspartate-64 and histidine-34 is not u nusually strong. The hydrogen bonding network linking tyrosine-87 to a spartate-64 and aspartate-64 to histidine-34 is not critical for catal ysis.