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 KI = 65 microM and a limiting k(inact) of 0.073 min-1 at 23 degrees 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.
Hruz
P W
PW
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Narasimhan
C
C
Miziorko
H M
HM
D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine; D010450:Endopeptidases; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011549:Pseudomonas / Q000201:enzymology*
D000214:Acyl Coenzyme A; D000345:Affinity Labels; D010446:Peptide Fragments; C075847:2-butynoyl-coenzyme A; D010450:Endopeptidases; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D003545:Cysteine