C. Narasimhan et al., PSEUDOMONAS MEVALONII 3-HYDROXY-3-METHYLGLUTARYL-COA LYASE - TESTING THE FUNCTION OF THE ACTIVE-SITE CYSTEINE BY SITE-DIRECTED MUTAGENESIS, Biochemistry, 34(31), 1995, pp. 9930-9935
In order to evaluate whether C237 functions in the chemistry of hydrox
ymethylglutaryl-CoA cleavage, cassette mutagenesis has been employed t
o alter wild-type DNA to encode serine or alanine at residue 237, ESR
measurements indicate that the purified mutant enzymes bind stoichiome
tric amounts of the spin-labeled substrate analog, R(.)CoA, which has
been established as a competitive inhibitor. Binding affinities measur
ed with C237S (K-d = 92 mu M) and C237A (K-d = 97 mu M) lyases are com
parable to that observed with wild-type lyase. The rotational dynamics
of R(.)CoA bound to mutant enzymes are also very similar to those for
R(.)CoA bound to wild-type lyase. These observations suggest that the
mutant enzymes are structurally intact. In view of this demonstrated
structural integrity, it is significant that the V(max)s of C237A and
C237S are approximate to 4 x 10(4)- and approximate to 725-fold lower,
respectively, than the value measured for wild-type hydroxymethylglut
aryl-CoA lyase, The C237S enzyme exhibits a K-m = 53 mu M for substrat
e; this value is only 2-fold higher than the K-m of the wild-type enzy
me. Additionally, we report that the residual activity in C237S hydrox
ymethylglutaryl-CoA lyase is unaffected by 2-butynoyl-CoA under condit
ions which support inactivation of wild-type enzyme. These results are
consistent with an active site assignment to C237, confirming the pre
diction based on the affinity labeling/peptide mapping data. Moreover,
they suggest that C237 is a crucial residue in the catalytic apparatu
s and qualify it for consideration as the active site residue that dep
rotonates the 3-hydroxyl group of hydroxymethylglutaryl-CoA.