PMID:10353839

Citation	Joyce, MA, Fraser, ME, Brownie, ER, James, MN, Bridger, WA and Wolodko, WT (1999) Probing the nucleotide-binding site of Escherichia coli succinyl-CoA synthetase. Biochemistry 38:7273-83
Abstract	Succinyl-CoA synthetase (SCS) catalyzes the reversible interchange of purine nucleoside diphosphate, succinyl-CoA, and Pi with purine nucleoside triphosphate, succinate, and CoA via a phosphorylated histidine (H246alpha) intermediate. Two potential nucleotide-binding sites were predicted in the beta-subunit, and have been differentiated by photoaffinity labeling with 8-N3-ATP and by site-directed mutagenesis. It was demonstrated that 8-N3-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-N3-ATP. These corresponded to residues 107-119beta and 121-146beta, two regions lying along one side of an ATP-grasp fold. A mutant protein with changes on the opposite side of the fold (G53betaV/R54betaE) was unable to be phosphorylated using ATP or GTP, but could be phosphorylated by succinyl-CoA and Pi. A mutant protein designed to probe nucleotide specificity (P20betaQ) had a Km(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 (I322betaA, and R324betaN/D326betaA) 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 approximately 35 A apart, and that the H246alpha loop moves between them during catalysis.
Links	PubMed Online version:10.1021/bi990527s
Keywords	Adenosine Diphosphate/chemistry; Adenosine Diphosphate/metabolism; Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/chemistry; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Azides/chemistry; Azides/metabolism; Binding Sites; Conserved Sequence; Enzyme Activation; Escherichia coli/enzymology; Guanosine Triphosphate/chemistry; Guanosine Triphosphate/metabolism; Mass Spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments/genetics; Peptide Fragments/isolation & purification; Phosphorylation; Photoaffinity Labels/chemistry; Photoaffinity Labels/metabolism; Purine Nucleotides/chemistry; Purine Nucleotides/metabolism; Substrate Specificity; Succinate-CoA Ligases/antagonists & inhibitors; Succinate-CoA Ligases/chemistry; Succinate-CoA Ligases/genetics; Succinate-CoA Ligases/metabolism
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