A comparison of acylation, phosphorylation and binding in related substrates and inhibitors of serum cholinesterase.

1. The K(m) and catalytic-centre activities for human serum cholinesterase and methyl, ethyl, n-propyl and n-butyl butyrate substrates were determined and compared with the related inhibition constants of a similarly substituted organophosphate inhibitor series based on malaoxon. The results indicated that the catalytic-centre activities approximated to k(+2(a)), the acylation rate constant, and that K(m) approximated to the equilibrium binding constant. The inhibition constants measured were K(a), the equilibrium binding constant, and k(+2(p)), the phosphorylation rate constant. 2. The effects of the alkyl substituents on k(+2(p)) and k(+2(a)) were closely parallel, and the decreasing order in each case was: n-butyl; methyl; n-propyl; ethyl. The Taft constants did not follow this order, suggesting that alkyl substituents did not primarily effect acylation or phosphorylation by electron induction. 3. For comparable homologues, the k(+2(a)) values were on average 435 times the k(+2(p)) values. The k(+2(p)) values at 25 degrees and pH7.6 ranged from 6.6min.(-1) for the diethyl member to 22.6min.(-1) for the di-n-butyl member. 4. The effect of the alkyl substituents on K(a) and K(m) were closely paralleled. The increasing order in each case was: n-butyl; n-propyl; ethyl; methyl. The K(a) values were about 100 times less than the comparable K(m) values. 5. Consideration of the binding energies suggested that only one of the two alkyl groups on the malaoxon homologues bound to the active site. 6. The possibility that malaoxon acted as a substrate as well as an inhibitor for cholinesterase was also investigated, but no evidence of a substrate reaction was found.