The Enzymatic Carboxylation of Phosphoenolpyruvate: IV. The binding of manganese and substrates by phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase

Abstract

Abstract The binding of Mn2+ and substrates to phosphoenolpyruvate carboxykinase has been studied by four methods: equilibrium dialysis, gel filtration, electron paramagnetic resonance, and the proton relaxation rate of water (PRR). A binary enzyme-Mn2+ complex was detected by electron paramagnetic resonance and PRR. Binary enzyme-substrate complexes were detected for guanosine triphosphate and guanosine diphosphate by equilibrium dialysis and for inosine diphosphate by gel filtration. Ternary complexes containing enzyme, Mn2+, and substrate were detected for IDP by gel filtration and PRR, for phosphoenolpyruvate by equilibrium dialysis and PRR, and for inosine triphosphate and oxalacetate by PRR. Higher order complexes of the form eMn-sMn were detected for GDP and GTP by equilibrium dialysis. Phospholactate, an analogue of phosphoenolpyruvate, formed ternary complexes with enzyme and Mn2+ and a stable quaternary complex with enzyme, Mn2+, and IDP, as detected by PRR. Where comparable, the dissociation constants obtained by the various methods were in good agreement. The enzyme enhanced the effect of Mn2+ on the PRR of water (eb = 14.2), and this enhancement was reduced in the ternary complexes with phosphoenolpyruvate (et = 6.7) and with IDP (et = 10.4). Since the relaxation mechanism was the same for each of these complexes (measuring the rate of chemical exchange of water protons into the coordination sphere of Mn2+), the relative values of e are consistent with the formation of enzyme-metal-substrate bridge complexes. The related enzyme, phosphoenolpyruvate carboxylase, gives similar results, forming a binary enzyme-Mn2+ complex (eb = 13.8) and a ternary complex with phosphoenolpyruvate (et ≤ 11.3). The Kd of the binary complex agrees with the activator constant for Mn2+ obtained kinetically. The complexes formed by both carboxylating enzymes are thus similar to those found previously for muscle pyruvate kinase and, therefore, suggest homologous mechanisms for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, and pyruvate kinase.