Subbaramaiah K, Simms S A
Department of Chemistry, City College, City University of New York, New York 10031.
J Biol Chem. 1992 Apr 25;267(12):8636-42.
CheR methyltransferase from Salmonella typhimurium was directly photolabeled with S-adenosyl-L-[methyl-3H]methionine. The labeled protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then was detected by fluorography. The methylase-S-adenosyl-L-methionine adduct was found to be stable under the experimental conditions employed. Labeling was found to be a function of the concentration of enzyme, S-adenosyl-L-methionine (AdoMet), and the intensity and time of UV irradiation. The extent of labeling and protein methylation was found to be inhibited by S-adenosyl-L-homocysteine, S-adenosyl-L-ethionine, and sinefungin, which are known to compete with AdoMet for the same binding site on the enzyme. Our earlier data showed that the enzyme has 2 cysteine residues and that these are important for enzyme activity. Here, we show that sulfhydryl reagents inhibit the photolabeling of the substrate to the enzyme, indicating the presence of cysteine in the vicinity of the substrate-binding site. We also found that when Cys31 was modified to Ser, no photolabeling of CheR was observed, whereas a modification of Cys229 to Ser had little effect on the ability of AdoMet to label the enzyme. This suggests that Cys31 is located at or near AdoMet-binding site. The labeled protein was cleaved at tryptophan residues, generating two major fragments, each containing 1 cysteine residue. SDS-PAGE and fluorography of the cleaved products indicated the presence of the label being associated with the Cys31 fragment. Similar results were obtained when the labeled protein was cleaved at glutamic acid residues using V8 protease. A tryptic digest of the labeled protein showed two radioactive peptide peaks when subjected to separation on reverse phase high pressure liquid chromatography. The labeled peptides were further digested to free amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that Cys31 may be involved with substrate binding, as well as with catalysis.
来自鼠伤寒沙门氏菌的CheR甲基转移酶用S-腺苷-L-[甲基-³H]甲硫氨酸直接进行光标记。将标记的蛋白质进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），然后通过荧光自显影进行检测。发现甲基化酶-S-腺苷-L-甲硫氨酸加合物在所采用的实验条件下是稳定的。发现标记是酶浓度、S-腺苷-L-甲硫氨酸（AdoMet）以及紫外线照射强度和时间的函数。已知S-腺苷-L-高半胱氨酸、S-腺苷-L-乙硫氨酸和杀稻瘟菌素与AdoMet竞争酶上的相同结合位点，发现它们会抑制标记程度和蛋白质甲基化。我们早期的数据表明该酶有2个半胱氨酸残基，并且这些残基对酶活性很重要。在此，我们表明巯基试剂会抑制底物与酶的光标记，这表明在底物结合位点附近存在半胱氨酸。我们还发现，当Cys31被修饰为Ser时，未观察到CheR的光标记，而将Cys229修饰为Ser对AdoMet标记酶的能力几乎没有影响。这表明Cys31位于AdoMet结合位点或其附近。标记的蛋白质在色氨酸残基处被切割，产生两个主要片段，每个片段含有1个半胱氨酸残基。切割产物的SDS-PAGE和荧光自显影表明标记与Cys31片段相关。当使用V8蛋白酶在谷氨酸残基处切割标记的蛋白质时，也获得了类似的结果。标记蛋白质的胰蛋白酶消化产物在反相高压液相色谱上分离时显示出两个放射性肽峰。标记的肽进一步消化为游离氨基酸，通过薄层色谱法将标记的氨基酸鉴定为S-甲基半胱氨酸。这些结果表明Cys31可能参与底物结合以及催化作用。
