Chemical events in chloropropionyl-coenzyme A inactivation of acyl-coenzyme A utilizing enzymes

Miziorko, Henry M.; Behnke, Christine E.; Ahmad, Feroz

doi:10.1021/bi00440a009

Cited by 8 publications

(11 citation statements)

References 32 publications

(46 reference statements)

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“…Work on yeast [42,43] and avian [44][45][46][47][48][49][50][51] mitochondrial HMG-CoA synthases established that the synthesis of HMG-CoA occurs in three steps. The first, which is rate-limiting, is the acetylation of the enzyme in a cysteinyl thiol group.…”

Section: Mechanism Of Enzyme Reactionmentioning

confidence: 99%

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis

Hegardt

1999

Biochemical Journal

293

218

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Cytosolic and mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthases were first recognized as different chemical entities in 1975, when they were purified and characterized by Lane's group. Since then, the two enzymes have been studied extensively, one as a control site of the cholesterol biosynthetic pathway and the other as an important control site of ketogenesis. This review describes some key developments over the last 25 years that have led to our current understanding of the physiology of mitochondrial HMG-CoA synthase in the HMG-CoA pathway and in ketogenesis in the liver and small intestine of suckling animals. The enzyme is regulated by two systems: succinylation and desuccinylation in the short term, and transcriptional regulation in the long term. Both control mechanisms are influenced by nutritional and hormonal factors, which explains the incidence of ketogenesis in diabetes and starvation, during intense lipolysis, and in the foetal-neonatal and suckling-weaning transitions. The DNA-binding properties of the peroxisome-proliferator-activated receptor and other transcription factors on the nuclear-receptor-responsive element of the mitochondrial HMG-CoA synthase promoter have revealed how ketogenesis can be regulated by fatty acids. Finally, the expression of mitochondrial HMG-CoA synthase in the gonads and the correction of auxotrophy for mevalonate in cells deficient in cytosolic HMG-CoA synthase suggest that the mitochondrial enzyme may play a role in cholesterogenesis in gonadal and other tissues.

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Section: Mechanism Of Enzyme Reactionmentioning

confidence: 99%

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis

Hegardt

1999

Biochemical Journal

293

218

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“…(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. After C-2 of the inhibitor is deprotonated, elimination of chloride and production of acryloylSCoA ensues.…”

Section: Comparison Of Catalysis Of Partial Reactions By Wild Type Anmentioning

confidence: 99%

3-Hydroxy-3-methylglutaryl-CoA Synthase

Chun¹,

Vinarov²,

Zajíček³

et al. 2000

Journal of Biological Chemistry

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Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acylCoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by 13 C NMR studies, which indicate that E95A forms an acetyl-S-enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu 95 functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK a of Glu 95 as required to support its function as a general acid.3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) 1 synthase catalyzes the committed step in ketogenic and cholesterogenic pathways. Early work (1, 2) on the purified enzyme identified reaction intermediates that indicate that catalysis of HMGCoA production involves a three-step process (Scheme 1).Recombinant forms of both the avian (3) and human (4) enzyme have been expressed in Escherichia coli and isolated at high levels of purity. 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. The functions of other active site residues that participate directly in catalysis have not yet been firmly established.The sensitivity of HMG-CoA synthase activity to treatment with a carboxyl-directed modification reagent led to a preliminary observation (9), which implicated Glu 95 as a residue that is important to reaction chemistry. This report documents the crucial role of Glu 95 in catalysis and indicates which of the three steps in the reaction is compromised upon replacement of the Glu 95 carboxyl. Finally, potential functions for Glu 95 are considered, and tests ai...

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“…Acryloyl-CoA has been suggested as the inactivating species during 3-chloropropionyl-CoA incubation with HMG-CoA synthase (58) and fatty acid synthase (59). In addition, S-acryloyl-N-acetylcysteamine was shown to inhibit HMG-CoA synthase and lyase and fatty acid synthase (60). However, the data with BTTB-, NPTB-, and DNPTB-CoA clearly show that multiple cycles of generation of this Michael acceptor within the active site of the dehydrogenase does not lead to significant inactivation.…”

Section: Acknowledgmentmentioning

confidence: 99%

Elimination Reactions in the Medium-Chain Acyl-CoA Dehydrogenase: Bioactivation of Cytotoxic 4-Thiaalkanoic Acids

et al. 1998

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A range of 4-thiaacyl-CoA derivatives has been synthesized to study the bioactivation of cytotoxic fatty acids by the mitochondrial medium-chain acyl-CoA dehydrogenase and the peroxisomal acyl-CoA oxidase. Both enzymes catalyze alpha-proton abstraction from normal acyl-CoA substrates with elimination of a beta-hydride equivalent to the FAD prosthetic group. In competition with this oxidation reaction, 4-thiaacyl-CoA thioesters undergo dehydrogenase-catalyzed beta-elimination, providing that the corresponding thiolates are sufficiently good leaving groups and can be accommodated by the active site of the enzyme. Thus, the dehydrogenase catalyzes the elimination of 2-mercaptobenzothiazole and 4-nitrothiophenolate from 4-(2-benzothiazole)-4-thiabutanoyl-CoA and 4-(4-nitrophenyl)-4-thiabutanoyl-CoA, respectively. However, the 2,4-dinitrophenyl-analogue appears too bulky and the unsubstituted thiophenyl-derivative is insufficiently activated for significant elimination. Molecular modeling shows that steric interference from the flavin ring dictates a syn rather than an anti elimination. Acryloyl-CoA, the other product of 4-thiaacyl-CoA elimination reactions, is not a significant inactivator of the medium-chain dehydrogenase. In contrast, the irreversible inactivation observed during beta-elimination using 5,6-dichloro-4-thia-5-hexenoyl-CoA (DCTH-CoA), 5,6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA), and 6-chloro-5,5,6-trifluoro-4-thiahexanoyl-CoA (CTFTH-CoA) is caused by release of cytotoxic thiolate products within the active site of the dehydrogenase. The double bond between C5 and C6 found in the vinylic analogues DCTH- and DCTFTH-CoA is not essential for enzyme inactivation, although CTFTH-CoA is a weaker inhibitor of the dehydrogenase. Mechanism-based inactivation with CTFTH-CoA requires elimination, is unaffected by exogenous nucleophiles, and is strongly protected by octanoyl-CoA. The peroxisomal acyl-CoA oxidase efficiently oxidizes 4-thiaacyl-CoA analogues, but is only rapidly inactivated by DCTFTH-CoA. The variable ratio of elimination to oxidation observed for DCTH-, DCTFTH-, and CTFTH-CoA may influence the metabolism of the corresponding cytotoxic alkanoic acids in vivo.

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Chemical events in chloropropionyl-coenzyme A inactivation of acyl-coenzyme A utilizing enzymes

Cited by 8 publications

References 32 publications

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis

3-Hydroxy-3-methylglutaryl-CoA Synthase

Elimination Reactions in the Medium-Chain Acyl-CoA Dehydrogenase: Bioactivation of Cytotoxic 4-Thiaalkanoic Acids

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