Probing the nucleotide-binding site of Escherichia coli succinyl-CoA synthetase.
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.
Joyce
M A
MA
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Fraser
M E
ME
Brownie
E R
ER
James
M N
MN
Bridger
W A
WA
Wolodko
W T
WT
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D017124:Conserved Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010766:Phosphorylation; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism; D011685:Purine Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013389:Succinate-CoA Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001386:Azides; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011685:Purine Nucleotides; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D013389:Succinate-CoA Ligases