3-Hydroxy-3-methylglutaryl Coenzyme A Lyase: Affinity Labeling of the Pseudomonas mevalonii Enzyme and Assignment of Cysteine-237 to the Active Site

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase is irreversibly inactivated by the reactive substrate analog 2-butynoyl-CoA. Enzyme inactivation, which follows pseudo-first-order kinetics, is saturable with a K_I = 65 µM and a limiting k_inact of 0.073 min^-1 at 23 °C, pH 7.2. Protection against inactivation is afforded by the competitive inhibitor 3-hydroxyglutaryl-CoA. Labeling of the bacterial enzyme with [1-¹⁴C] -2-butynoyl-CoA demonstrates that inactivation coincides with covalent incorporation of inhibitor, with an observed stoichiometry of modification of 0.65 per site. Avian HMG-CoA lyase is also irreversibly inactivated by 2-butynoyl-CoA with a stoichiometry of modification of 0.9 per site. Incubation of 2-butynoyl-CoA with mercaptans such as dithiothreitol results in the formation of a UV absorbance peak at 310 nm. Enzyme inactivation is also accompanied by the development of a UV absorbance peak at 310 nm indicating that 2-butynoyl-CoA modifies a cysteine residue in HMG-CoA lyase. Tryptic digestion and reverse-phase HPLC of the affinity-labeled protein reveal a single radiolabeled peptide. Isolation and sequence analysis of this peptide and a smaller chymotryptic peptide indicate that the radiolabeled residue is contained within the sequence GGXPY. Mapping of this peptide within the cDNA-deduced sequence of P. mevalonii HMG-CoA lyase [Anderson, D.H., & Rodwell, V.W. (1989) J. Bacteriol. 171, 6468-6472] confirms that a cysteine at position 237 is the site of modification. These data represent the first identification of an active-site residue in HMG-CoA lyase.