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真菌β-内酯hymeglusin在胞质3-羟基-3-甲基戊二酰辅酶A合酶上的结合位点。

Binding site for fungal beta-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase.

作者信息

Tomoda Hiroshi, Ohbayashi Naomi, Morikawa Yuko, Kumagai Hidetoshi, Omura Satoshi

机构信息

Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, and The Kitasato Institute, 5-9-1 Shirokane, Minato, Tokyo 108-8641, Japan.

出版信息

Biochim Biophys Acta. 2004 Feb 27;1636(1):22-8. doi: 10.1016/j.bbalip.2003.11.005.

Abstract

We studied the molecular mechanism through which the fungal beta-lactone, hymeglusin, potently and specifically inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. [(14)C]Hymeglusin covalently bound to purified rat liver and to recombinant hamster cytosolic HMG-CoA synthases. The enzyme activity was completely inhibited at a binding ratio of 1.6-2.0 mol [(14)C]hymeglusin/mol HMG-CoA synthase. Incubating the enzyme with 2 mM iodoacetamide (IAA) or 2 mM N-ethylmaleimide (NEM) but not with 1.0 mM diisopropyl fluorophosphates (DFP) completely inhibited the binding, suggesting that hymeglusin binds to a Cys residue of HMG-CoA synthase. Recombinant hamster HMG-CoA synthase labeled with [(3)H]hymeglusin was digested with V8 protease, and the [(3)H]peptide was purified by high performance liquid chromatography (HPLC). The sequence of the peptide was Ser-Gly-Asn-Thr-Asp-Ile-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-[(3)H]hymeglusyl Cys-Tyr-Gly-Gly-Thr-Ala-Ala-Val-Phe-Asn-Ala-Val-Asn-, which corresponds to the active site sequence (from Ser 115 to Asn 141) of hamster HMG-CoA synthase. These findings showed that hymeglusin inhibits hamster cytosolic HMG-CoA synthase by covalently modifying the active Cys 129 residue of the enzyme.

摘要

我们研究了真菌β-内酯hymeglusin有效且特异性抑制3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)合酶的分子机制。[¹⁴C]hymeglusin与纯化的大鼠肝脏和重组仓鼠胞质HMG-CoA合酶共价结合。当结合比例为1.6 - 2.0摩尔[¹⁴C]hymeglusin/摩尔HMG-CoA合酶时,酶活性被完全抑制。用2 mM碘乙酰胺(IAA)或2 mM N-乙基马来酰亚胺(NEM)而非1.0 mM二异丙基氟磷酸酯(DFP)孵育该酶,可完全抑制这种结合,这表明hymeglusin与HMG-CoA合酶的一个半胱氨酸残基结合。用V8蛋白酶消化标记有[³H]hymeglusin的重组仓鼠HMG-CoA合酶,并用高效液相色谱(HPLC)纯化[³H]肽段。该肽段的序列为Ser-Gly-Asn-Thr-Asp-Ile-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-[³H]hymeglusyl Cys-Tyr-Gly-Gly-Thr-Ala-Ala-Val-Phe-Asn-Ala-Val-Asn-,它对应于仓鼠HMG-CoA合酶的活性位点序列(从Ser 115到Asn 141)。这些发现表明,hymeglusin通过共价修饰该酶的活性半胱氨酸129残基来抑制仓鼠胞质HMG-CoA合酶。

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