PSEUDOMONAS MEVALONII 3-HYDROXY-3-METHYLGLUTARYL-COA LYASE - TESTING THE FUNCTION OF THE ACTIVE-SITE CYSTEINE BY SITE-DIRECTED MUTAGENESIS

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.