Inactivation studies of acetylcholinesterase with phenylmethylsulfonyl fluoride

Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsulfonyl fluoride (PMSF) and benzenesulfony l fluoride (BSF). Although BSF inhibits both mouse and Torpedo californica AChE, PMSF does not react measurably with the T. californica enzyme. To und erstand the residue changes responsible for the change in reactivity, we st udied the inactivation of wild-type T. californica and mouse AChE and mutan ts of both by BSF and PMSF both in the presence and absence of substrate. T he enzymes investigated were wild-type mouse AChE, wild-type T. californica AChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, an d F290I, and the double mutant T. californica F288L/F290V (all mutants give n T. californica numbering). Inactivation rate constants for T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMS F. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar i nactivation rate constants with PMSF, implying that the difference between mouse and T. californica AChE at position 330 is not responsible for their differing PMSF sensitivities. In addition, butyrylcholinesterase and mouse AChE mutants F288L and F290I had increased rate constants (similar to 14 fo ld) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation by PMSF of but yrylcholinesterase. The double mutant T. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F2 90I mouse mutant AChEs, representing an increase of similar to 4000-fold ov er the T. californica wild-type enzyme. It remains unclear why these two po sitions have more importance for T. californica AChE than for mouse AChE.