Organophosphate Specificity of Acyl Pocket Cholinesterase Mutants

Abstract

Organophosphate (OP) compounds potently and irreversibly inhibit cholinesterases by phosphorylating the active site serine. Their tetrahedral geometry about the phosphate, in contrast to the trigonal carbonyl carbon of carboxyl or carbamoyl ester substrates, adds another dimension to substituent substitution. Therefore, size and geometric constraints as well as stereospecificity can be examined. As reported (Berman & Leonard, 1989), Torpedo acetylcholinesterase (AChE) has marked stereospecificity and substituent size specificity to OP’s with the differences in reaction rates up to 200 fold between enantiomers. This specificity is suspected to be due to steric hindrance as found for substrate specificity between AChE and butyrylcholinesterase (BuChE) (Vellom et. al., 1993). The side chains of F295 and F297 have been shown in several studies to form the steric constraints on acyl pocket size, however, these residues will be shown to have distinct influences on substrate catalysis and substrate inhibition for carbonyl substrates. In the case of alkylphosphates, a more discriminating analysis of structure function is possible where we have examined acylation by the two enantiomers of a series of alkyl methyl phosphonyl thiocholines in relation to acyl pocket sustituents. Such studies provide a data base for modeling the orientation of substituent groups in the transition state and the acyl enzyme. (Supported by USHPHS grant GM 18360 & DAMD17-9-C1058).