Identification of the Cysteine Residue Exposed by the Conformational Change in Pig Heart Succinyl-CoA:3-Ketoacid Coenzyme A Transferase on Binding Coenzyme A

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• 作者：Stephanie D. Tammam ; Jean-Christophe Rochet ; Marie E. Fraser
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：September 25, 2007
• 年：2007
• 卷：46
• 期：38
• 页码：10852 - 10863
• 全文大小：543K
• 年卷期：v.46,no.38(September 25, 2007)
• ISSN：1520-4995

文摘

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA toacetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA islinked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoicacid), reflecting a conformational change of SCOT upon formation of the thioester. Since either Cys 28or Cys 196 could be the target, each was mutated to Ser to distinguish between them. Like wild-typeSCOT, the C196S mutant protein was modified rapidly in the presence of acyl-CoA substrates. In contrast,the C28S mutant protein was modified much more slowly under identical conditions, indicating that Cys28 is the residue exposed on binding CoA. The specific activity of the C28S mutant protein wasunexpectedly lower than that of wild-type SCOT. X-ray crystallography revealed that Ser adopts a differentconformation than the native Cys. A chloride ion is bound to one of four active sites in the crystal structureof the C28S mutant protein, mimicking substrate, interacting with Lys 329, Asn 51, and Asn 52. On thebasis of these results and the studies of the structurally similar CoA transferase from Escherichia coli,YdiF, bound to CoA, the conformational change in SCOT was deduced to be a domain rotation of 17coupled with movement of two loops: residues 321-329 that bury Cys 28 and interact with succinate oracetoacetate and residues 374-386 that interact with CoA. Modeling this conformational change has ledto the proposal of a new mechanism for catalysis by SCOT.