Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

Abstract

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue.

Mitochondrial protein thiol modifications in acetaminophen hepatotoxicity: Effect on HMG-CoA synthase

“…HMG-CoA synthase catalyzes the conversion of acetoacetyl-CoA and acetyl-CoA to HMG-CoA and CoA (Zschocke et al, 2002;Hegardt, 1999). Interestingly, HMG-CoA synthase contains one active site cysteine residue (Miziorko and Behnke, 1985) and mutation of this active site cysteine has been shown to eliminate HMGCoA synthase activity (Rokosz et al, 1994). Because HMG-CoA synthase protein level was unchanged in the current study following APAP exposure, it is proposed that the APAP-dependent decrease in HMG-CoA synthase activity ( Figure 5F) is due to oxidative modification of the cysteine residue in the active site of the enzyme.…”

3-Hydroxy-3-methylglutaryl-CoA Synthase

“…DNA sequencing was performed using an ALF automated sequencer, and the cyclosequencing kit and protocol were provided by Amersham Pharmacia Biotech. Ampicillin and isopropyl-␤-D-thiogalactoside were purchased from U.S. Biochemical Corp. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetyl-CoA was purchased from Moravek Biochemicals (Brea, CA) and 3-chloro- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]propionic acid from American Radiolabeled Chemicals (St. Louis, MO). All other reagents were purchased from Sigma, Aldrich, Amersham Pharmacia Biotech, or Bio-Rad.…”

“…Precipitated [1-14 C]acetyl-S-enzyme was loaded onto a glass fiber filter, washed, and counted. (5,6) has demonstrated that cysteine 129 is selectively alkylated by the substrate analog, 3-chloropropionyl-CoA. Additional studies (18) suggest that the process reflects a mechanism-based process, with a general base on the enzyme catalyzing deprotonation of C-2 of the analog's acyl group, a reaction comparable with the deprotonation step that precedes productive condensation between the enzyme's normal substrates.…”

“…The recombinant human enzyme has been used to demonstrate that HMG-CoA synthase is the target of a potent antisteroidogenic drug (4). Mutagenesis work on the recombinant avian enzyme (3) has confirmed that Cys 129 is required to form the acetyl-S-enzyme intermediate (Scheme 1), a hypothesis that was initially advanced on the basis of protein modification by a mechanism-based inhibitor (5,6) as well as sequence analysis of a peptide that harbors the acetyl-S-Cys 129 adduct (7). 2 Additionally, kinetic characterization of mutants in which His 264 has been replaced implicates that residue (8) in binding of the second substrate, acetoacetyl-CoA.…”

Mitochondrial HMG–CoA Synthase Deficiency

“…At the N-terminal end is the leader peptide, with 37 aa, which drives the protein to the mitochondria. In 1985, the catalytic sequence of the enzyme was identified in a purified protein from chicken liver (Miziorko & Behnke, 1985). This sequence has 21 aa and a 100% homology with HMG-CoA synthases from other mammals.…”