Abstract

Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsulfonyl fluoride (PMSF) and benzenesulfonyl fluoride (BSF). Although BSF inhibits both mouse andTorpedo californica AChE, PMSF does not react measurably with the T.californica enzyme. To understand the residue changes responsible for the change in reactivity, we studied the inactivation of wild-type T. californica and mouse AChE and mutants of both by BSF and PMSF both in the presence and absence of substrate. The enzymes investigated were wild-type mouse AChE, wild-type T. californicaAChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, and F290I, and the double mutant T. californicaF288L/F290V (all mutants given T. californicanumbering). Inactivation rate constants for T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMSF. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar inactivation 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 (∼14 fold) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation by PMSF of butyrylcholinesterase. The double mutantT. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F290I mouse mutant AChEs, representing an increase of ∼4000-fold over theT. californica wild-type enzyme. It remains unclear why these two positions have more importance for T. californica AChE than for mouse AChE.