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

Succinyl-CoA synthetase (SCS) catalyzes the reversible interchange of purin e nucleoside diphosphate, succinyl-CoA, and P-i with purine nucleoside trip hosphate. succinate, and CoA via a phosphorylated histidine (H246 alpha) in termediate. Two potential nucleotide-binding sites were predicted in the be ta-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 label ed 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 prote in with changes on the opposite side of the fold (G53 beta V/R53 beta E) wa s 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 spe cificity (P20 beta Q) had a K-m(app) for GTP that was more than 5 times low er than that of wild-type SCS, whereas parameters for the other substrates remained unchanged. Mutations of residues in the C-terminal domain of the b eta-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 su ccinate and CoA. They did not disrupt the phosphorylation of SCS with nucle otides. 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 act ive sites similar to 35 Angstrom apart, and that the H246 alpha loop moves between them during catalysis.