Probing the nucleotide-binding site of Escherichia coli succinyl-CoA synthetase

被引:29
作者
Joyce, MA
Fraser, ME
Brownie, ER
James, MNG
Bridger, WA
Wolodko, WT [1 ]
机构
[1] Univ Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
[2] Univ Alberta, MTC Canada, Grp Prot Struct & Funct, Edmonton, AB T6G 2H7, Canada
[3] Univ Western Ontario, Off VP Res, London, ON N6A 5B8, Canada
关键词
D O I
10.1021/bi990527s
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Succinyl-CoA synthetase (SCS) catalyzes the reversible interchange of purine nucleoside diphosphate, succinyl-CoA, and P-i with purine nucleoside triphosphate. succinate, and CoA via a phosphorylated histidine (H246 alpha) intermediate. Two potential nucleotide-binding sites were predicted in the beta-subunit, and have been differentiated by photoaffinity labeling with 8-N-3-ATP and by site-directed mutagenesis. It was demonstrated that 8-N-3-ATP is a suitable analogue for probing the nucleotide-binding site of SCS. Two tryptic peptides from the N-terminal domain of the beta-subunit were labeled with 8-N-3-ATP. These corresponded to residues 107-119 beta and 121-146 beta, two regions lying along one side of an ATP-grasp Fold. A mutant protein with changes on the opposite side of the fold (G53 beta V/R53 beta E) was unable to be phosphorylated using ATP or GTP, but could be phosphorylated by succinyl-CoA and P-i. A mutant protein designed to probe nucleotide specificity (P20 beta Q) had a K-m(app) for GTP that was more than 5 times lower than that of wild-type SCS, whereas parameters for the other substrates remained unchanged. Mutations of residues in the C-terminal domain of the beta-subunit designed to distrupt one loop of the Rossmann fold (I322 beta A, and R324 beta N/D326 beta A) had the greatest effect on the binding of succinate and CoA. They did not disrupt the phosphorylation of SCS with nucleotides. It was concluded that the nucleotide-binding site is located in the N-terminal domain of the beta-subunit. This implies that there are two active sites similar to 35 Angstrom apart, and that the H246 alpha loop moves between them during catalysis.
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收藏
页码:7273 / 7283
页数:11
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