ADP-binding site of Escherichia coli succinyl-CoA synthetase revealed by X-ray crystallography

Succinyl-CoA synthetase (SCS) catalyzes the following reversible reaction v ia a phosphorylated histidine intermediate (His 246 alpha): succinyl-CoA P-i + NDP <-> succinate + CoA + NTP (N denotes adenosine or guanosine). To determine the structure of the enzyme with nucleotide bound, crystals of ph osphorylated Escherichia coli SCS were soaked in successive experiments ado pting progressive strategies. In the first experiment, 1 mM ADP (>15 x K-d) was added; Mg2+ ions were omitted to preclude the formation of an insolubl e precipitate with the phosphate and ammonium ions. X-ray crystallography r evealed that the enzyme was dephosphorylated, but the nucleotide did not re main bound to the enzyme (R-working = 17.2%, R-free = 22.8% for data to 2.9 Angstrom resolution). Catalysis requires Mg2+ ions; hence, the "true" nucl eotide substrate is probably an ADP-Mg2+ complex. In the successful experim ent, the phosphate buffer was exchanged with MOPS, the concentration of sul fate ions was lowered, and the concentrations of ADP and Mg2+ ions were inc reased to 10.5 and 50 mM, respectively. X-ray diffraction data revealed an ADP-Mg2+ complex bound in the ATP-grasp fold of the N-terminal domain of ea ch beta-subunit (R-working = 19.1%, R-free = 24.7% for data to 3.3 Angstrom resolution). We describe the specific interactions of the nucleotide-Mg2complex with SCS, compare these results with those for other proteins conta ining the ATP-grasp fold, and present a hypothetical model of the histidine -containing loop in the "down" position where it can interact with the nucl eotide similar to 35 Angstrom from where His 246a is seen in both phosphory lated and dephosphorylated SCS.