Role of inosine 5'-phosphate in activating glucose-bisphosphatase.
Glucose-bisphosphate (G1c-1,6-P2) phosphatase has been purified greater than 200-fold from the cytosol of mouse brain. As reported earlier, the enzyme requires inosine monophosphate (IMP) and Mg2+ for activity [Guha, S.K., & Rose, Z. B. (1982) J. Biol. Chem. 257, 6634-6637]. Kinetic parameters and the role of IMP have been further investigated. When Glc-1,6-P2 and IMP are both varied, double-reciprocal plots of the data form a parallel line pattern. With 2 mM Mg2+, the Km obtained for G1c-1,6-P2 is 20 microM and the Ka for IMP is 9 microM. Co2+, Mn2+, and Ni2+ activate less effectively than Mg2+. The apparent Ka for Mg2+ decreases with increasing G1c-1,6-P2, and the observed Km of G1c-1,6-P2 decreases with increasing Mg2+. The extrapolated value of the Ka of Mg2+ at infinite substrate is 86 microM. Mg2+ does not affect the Ka of IMP. The phosphatase activity is optimal at pH 7. The phosphatase is not completely specific since mannose 1,6-bisphosphate is hydrolyzed and guanosine monophosphate activates. However, fructose 1,6-bisphosphate is no more than a poor inhibitor, and adenine nucleotides are neither activators nor inhibitors. The products of the reaction are glucose-1-P and glucose-6-P, in a ratio of 2:3, and Pi. Both glucose-P's are competitive inhibitors with respect to IMP [Ki(glucose-1-P) = 5 microM; Ki(glucose-6-P) = 18 microM]. Neither glucose-P competes with G1c-1,6-P2. The demonstration of an exchange reaction between G1c-1,6-P2 and glucose-6-P is evidence for the phosphorylation of the enzyme by the substrate. The exchange reaction requires Mg2+ and is inhibited by IMP. The observation of the exchange reaction and its elimination by IMP indicates that the low level of phosphoglucomutase activity that remains with the phosphatase throughout purification is an inherent property of the phosphatase. The requirement of glucose-bisphosphatase for the nucleotide IMP is consistent with possible roles for both G1c-1,6-P2 and IMP in the control of the ATP level in the brain.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002413:Cations, Divalent; D002845:Chromatography / Q000379:methods; D004789:Enzyme Activation; D007291:Inosine Monophosphate / Q000502:physiology*; D007292:Inosine Nucleotides / Q000502:physiology*; D007700:Kinetics; D008297:Male; D008670:Metals; D051379:Mice; D008813:Mice, Inbred ICR; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002413:Cations, Divalent; D007292:Inosine Nucleotides; D008670:Metals; D013439:Sulfhydryl Reagents; D007291:Inosine Monophosphate; C014818:glucose 1,6-diphosphate phosphatase; D010744:Phosphoric Monoester Hydrolases