Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviou
r showing substrate activation with butyrylthiocholine (ETC) as the substra
te. The D70G mutant has a catalytic constant identical to that of the wild-
type enzyme, but a IO-fold lower affinity for ETC compared to wild-type enz
yme, and it does not exhibit activation by excess ETC under conventional co
nditions. In the present work it was found that addition of polyols or suga
rs changed the kinetic behaviour of the D70G mutant with ETC. In the presen
ce of 40% sucrose, the D70G mutant enzyme displayed marked activation by ex
cess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 wer
e studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE,
whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activa
tion. The behaviour of wild-type, Y332F, Y332A and Y332A/D70G did not chang
e in the presence of high concentrations of sugar. Substrate activation has
been explained by binding of a second substrate molecule in the peripheral
site at D70. The D70G mutant should be incapable of substrate activation,
if D70 were the only residue involved in substrate activation. The ability
of the D70G mutant to display substrate activation by medium engineering su
ggests that other residues are involved in initial substrate binding and ac
tivation by excess substrate. Osmolyte-induced change in conformation and/o
r hydration status of Y332 and other solvent-exposed residues may account f
or the non-Michaelian behaviour of the D70G mutant. (C) 1999 Elsevier Scien
ce B.V. All rights reserved.