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24-C-甾醇甲基转移酶的光亲和标记与突变分析确定了腺苷甲硫氨酸结合位点。

Photoaffinity labeling and mutational analysis of 24-C-sterol methyltransferase defines the AdoMet binding site.

作者信息

Jayasimha Pruthvi, Nes W David

机构信息

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.

出版信息

Lipids. 2008 Aug;43(8):681-93. doi: 10.1007/s11745-008-3198-x. Epub 2008 Jun 18.

Abstract

Photolabeling and site-directed mutagenesis were performed on recombinant Saccharomyces cerevisiae 24-C-sterol methyltransferase (SMT) to elucidate the location and role of active site residues involved in AdoMet binding and catalysis. Bioinformatic analysis of the SMT revealed a ten amino acid segment, conserved between L124 and P133, associated with the Rossmann-like fold belonging to AdoMet-dependent methyltransferases. Irradiation of the SMT in the presence of [methyl-3H3]AdoMet directly photolabeled the protein. The specificity of photolabeling was demonstrated by inactivation experiments with structural analogs of AdoMet, including sinefungin. Trypsin digestion of the [methyl-3H3]AdoMet photolabeled Erg6p afforded a single radioactive band in SDS-PAGE gel of 4 kDa. HPLC purification of this material generated a single radioactive fraction. The corresponding 3H-AdoMet-peptide adduct was subjected to Edman sequencing and the first fifteen residues gave a sequence Gly120-Asp-Leu-Val-Leu-Asp-Val-Gly-Cys-Gly-Val-Gly-Gly-Pro-Ala134 that contained the predicted AdoMet binding site. Amino acid residues in the tryptic digest fragment considered to bind covalently with the AdoMet at Asp125, Cys128, Pro133 and Tyr153 were replaced with leucine and analyzed kinetically and by photolabeling inactivation experiments. The results indicate that one or both of Cys128 and Pro133 are covalently bound to AdoMet.

摘要

对重组酿酒酵母24-C-甾醇甲基转移酶(SMT)进行了光标记和定点诱变,以阐明参与腺苷甲硫氨酸(AdoMet)结合和催化的活性位点残基的位置和作用。对SMT的生物信息学分析揭示了一个在L124和P133之间保守的十个氨基酸片段,与属于依赖AdoMet的甲基转移酶的类罗斯曼折叠相关。在[甲基-³H₃]AdoMet存在下对SMT进行辐照可直接对该蛋白质进行光标记。用包括杀稻瘟菌素在内的AdoMet结构类似物进行失活实验,证明了光标记的特异性。用胰蛋白酶消化[甲基-³H₃]AdoMet光标记的Erg6p,在SDS-PAGE凝胶上产生了一条4 kDa的单一放射性条带。对该物质进行HPLC纯化得到一个单一的放射性馏分。对相应的³H-AdoMet-肽加合物进行埃德曼测序,前十五个残基给出的序列为Gly120-Asp-Leu-Val-Leu-Asp-Val-Gly-Cys-Gly-Val-Gly-Gly-Pro-Ala134,其中包含预测的AdoMet结合位点。将胰蛋白酶消化片段中被认为与AdoMet共价结合的天冬氨酸125、半胱氨酸128、脯氨酸133和酪氨酸153处的氨基酸残基替换为亮氨酸,并进行动力学分析和光标记失活实验。结果表明,半胱氨酸128和脯氨酸133中的一个或两个与AdoMet共价结合。

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