3-HYDROXY-3-METHYLGLUTARYL-COA LYASE - EXPRESSION AND ISOLATION OF THE RECOMBINANT HUMAN ENZYME AND INVESTIGATION OF A MECHANISM FOR REGULATION OF ENZYME-ACTIVITY
Jr. Roberts et al., 3-HYDROXY-3-METHYLGLUTARYL-COA LYASE - EXPRESSION AND ISOLATION OF THE RECOMBINANT HUMAN ENZYME AND INVESTIGATION OF A MECHANISM FOR REGULATION OF ENZYME-ACTIVITY, The Journal of biological chemistry, 269(27), 1994, pp. 17841-17846
cDNA encoding the mature form of human hydroxymethylglutaryl-CoA (HMG-
CoA) lyase, a mitochondrial matrix protein, has been used to prepare e
xpression plasmids appropriate for production of this protein in Esche
richia coli. Using a T7 RNA polymerase-based pET system, HMG-CoA lyase
was overexpressed but largely recovered in an insoluble, catalyticall
y inactive form. In contrast, an expression plasmid (pTrcHL-1), derive
d from pTrc99a, supported production of soluble, active enzyme. A synt
hetic oligonucleotide cassette was employed to produce an enzyme varia
nt in which cysteine was replaced by serine at position 323. Both wild
-type and C323S HMG-CoA lyases were isolated in homogeneous form and c
haracterized. The function of Cys-323 in influencing catalytic activit
y in vitro has been investigated by comparing the response of wild-typ
e and C323S lyases to oxidation and reduction. Additionally, the con s
equences of treatment of these enzymes with the sulfhydryl-directed bi
functional reagent, o-phenylenedimaleimide have been determined. The r
esults support the hypothesis that a thiol/disulfide exchange mechanis
m affects enzyme activity in vitro and indicate that Cys-323 residues
on adjacent subunits of the homodimeric native enzyme are suitably pos
itioned to form an intersubunit cross-link upon oxidative inactivation
and disulfide formation.