Jr. Roberts et al., EVALUATION OF CYSTEINE-266 OF HUMAN 3-HYDROXY-3-METHYLGLUTARYL-COA LYASE AS A CATALYTIC RESIDUE, The Journal of biological chemistry, 270(29), 1995, pp. 17311-17316
The role of cysteine 266 in human 3-hydroxy-3-methylglutaryl-CoA (HMG
CoA) lyase, a residue that is homologous to a cysteine mapped to the a
ctive site of prokaryotic HMG-CoA lyase by protein chemistry approache
s, has been investigated by site-directed mutagenesis. Both the wild-t
ype human enzyme and a C323S variant, in which a regulatory sulfhydryl
has been eliminated without any negative effect on catalytic activity
(Roberts, J. R., Narasimhan, C., Hruz, P. W., Mitchell, G. A. and Miz
iorko, H. M. (1994) J. Biol. Chem. 269, 17841-17846), were used as mod
els. Mutant enzymes C266A, C266A/C323S, C266S, and C266S/C323S were ov
erexpressed in Escherichia coli and purified to homo geneity. In all c
ases, kinetic characterization indicated that the K-m value for HMG-Co
A was not substantially different from the value measured using wild-t
ype human lyase, suggesting that no serious structural perturbation oc
curs upon replacing Cys-266. A dissociable divalent cation (Mn2+ or Mg
2+), which is required for activity in both native and C323S enzymes,
is also an essential component for activity in each of the Cys-266 mut
ants. The structural integrity of the human mutants was further indica
ted by Mn2+ binding studies, which demonstrate similarities not only i
n the activator cation binding stoichiometries, but also in the K-D va
lues for Mn2+ as determined for wild-type and mutant C266A or C266S pr
oteins. Purified C266A and C266A/C323S mutants both displayed approxim
ate to 1.3 x 10(4)-fold diminution in specific activity, while the k(c
at) value was diminished in both C266S and C266S/C323S by approximate
to 9.9 x 10(2) fold. This large diminution in catalytic efficiency in
enzyme variants that display no substantial structural perturbations i
s in accord with an active site assignment to Cys-266 and qualifies it
s sulfhydryl group for consideration as a component of the catalytic a
pparatus.