P. Masson et al., ROLE OF ASPARTATE-70 AND TRYPTOPHAN-82 IN BINDING OF SUCCINYLDITHIOCHOLINE TO HUMAN BUTYRYLCHOLINESTERASE, Biochemistry, 36(8), 1997, pp. 2266-2277
The atypical Variant of human butyrylcholinesterase has Gly in place o
f Asp 70, Patients with this D70G mutation respond abnormally to the m
uscle relaxant succinyldicholine, experiencing hours of apnea rather t
han the intended 3 min. Asp 70 is at the rim of the active site gorge
12 A from the active site Ser 198. An unanswered question in the Liter
ature is why the atypical variant has a 10-fold increase in K-m for co
mpounds with a single positive charge but a 100-fold increase in K-m f
or compounds with two positive charges. We mutated residues Asp 70, Tr
p 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in ma
mmalian cells. Steady-state kinetic parameters far hydrolysis of butyr
lthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl
butyrate were determined. The wild type and the D70G mutant had ident
ical k(cat) values for all substrates. Molecular modeling and molecula
r dynamics suggested that succinyldicholine could bind in two consecut
ive orientations in the active site gorge, formation of one complex ca
used a conformational change in the omega loop involving Asp 70 and Tr
p 82. We propose the formation of three enzyme-substrate intermediates
preceding the acyl-enzyme intermediate; kinetic data support this con
tention, Substrates with a single positive charge interact with Asp 70
just once, whereas substrates with two positive charges, for example
succinyldithiocholine, interact with Asp 70 in two complexes, thus exp
laining the 10- and 100-fold increases in K-m in the D70G mutant.