Phosphoenolpyruvate carboxykinase. Mn2+ and Mn2+ substrate complexes.

Avian liver phosphoenolpyruvate carboxykinase was shown to have one binding site for Mn2+ by EPR and proton longitudinal relaxation rate measurements.The binary enzyme Mn complex has a water proton relaxation enhancement value of 12.2 f 1.7 and a K d = 5.0 -+ 0.5 X lo-' M. The Mn2+ binds more tightly in the presence of P-enolpyruvate (9.2 f 2.0 X IO-' M).Enzyme with low specific activity in the absence of mercaptan requires the presence of dithiothreitol to obtain maximum Mn2+ binding.The enzyme Mn complex binds phosphoenolpyruvate with a Kd = 0.6 f 0.4 X lo-' M and elicits an et = 6.6.The data suggest a substantial environmental change at the bound M n 2 + by this substrate.The HC03interacts with the enzyme-Mn complex with a & = 25 f 2 ~l l ~ and yields an et = 22.7.These results suggest that this substrate also elicits a substantial environmental change about the bound Mn2', but different from the change induced by phosphoenolpyruvate.The IDP interacts with a K d = 7.5 f 2.5 X lo-' M and yields an et = 13.8, suggesting no direct interaction with the bound Mn2+.ITP which binds with a K d = 3.8 f 1.1 X 10" M elicits an et = 6.2.These results suggest that the phosphoryl group which undergoes transfer during the catalytic reaction interacts directly with the bound M n 2 + .Each combination of quaternary enzyme *Mn SI Sz complexes has been detected by proton longitudinal relaxation rate titration studies.The results suggest that the active quinary complex can form by a random addition of substrates.These results suggest differences in the active site structures of the enzyme from pig liver (Miller, R.