Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: testing the function of the active site cysteine by site-directed mutagenesis.
Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase is affinity labeled by 2-butynoyl-CoA; peptide sequence analysis demonstrates C237 to be the site of modification [Hruz et al. (1992) Biochemistry 31, 6842-6847]. In order to evaluate whether C237 functions in the chemistry of hydroxymethylglutaryl-CoA cleavage, cassette mutagenesis has been employed to alter wild-type DNA to encode serine or alanine at residue 237. ESR measurements indicate that the purified mutant enzymes bind stoichiometric amounts of the spin-labeled substrate analog, R.CoA, which has been established as a competitive inhibitor. Binding affinities measured with C237S (Kd = 92 microM) and C237A (Kd = 97 microM) lyases are comparable 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 VmaxS of C237A and C237S are approximately 4 x 10(4)- and approximately 725-fold lower, respectively, than the value measured for wild-type hydroxymethylglutaryl-CoA lyase. The C237S enzyme exhibits a Km = 53 microM for substrate; this value is only 2-fold higher than the Km of the wild-type enzyme. Additionally, we report that the residual activity in C237S hydroxymethylglutaryl-CoA lyase is unaffected by 2-butynoyl-CoA under conditions which support inactivation of wild-type enzyme. These results are consistent with an active site assignment to C237, confirming the prediction based on the affinity labeling/peptide mapping data.(ABSTRACT TRUNCATED AT 250 WORDS)
Narasimhan
C
C
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Roberts
J R
JR
Miziorko
H M
HM
D000214:Acyl Coenzyme A / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013113:Spin Labels
D000214:Acyl Coenzyme A; D013113:Spin Labels; C075847:2-butynoyl-coenzyme A; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D003545:Cysteine