Studies on Phosphomannose Isomerase

Phosphomannose isomerase, which has previously been shown to be a zinc-metalloenzyme (J. Biol. Chem., 243, 4109 (1968)), was studied with respect to the following points: (a) effect of substrate on inhibition by metal-binding agents, (b) effect of zinc on the nonenzymatic isomerization, (c) effect of pH on Michaelis constant and maximal velocity, and (d) affinity of substrate analogues acting as competitive inhibitors. The substrate mannose 6-phosphate was found to protect phosphomannose isomerase from inhibition by o-phenanthroline, ethylenediaminetetraacetate, or dithiothreitol, suggesting that the metal is located in the active center of the enzyme. Zinc was found also to accelerate the nonenzymatic conversion of mannose 6-phosphate to fructose 6-phosphate and is believed to participate in a similar manner in both the nonenzymatic and the enzymatically catalyzed isomerization reactions. From the pH dependence of the kinetic parameters, pK values of 6.6 and 7.8 for the free enzyme and of 6.4 and 8.1 for the enzyme-substrate complex have been determined for two ionizable groups involved in substrate binding and catalysis; the pK values of 6.6 and 6.4 have been assigned to the imidazole group of a histidine residue. Analysis of the inhibition by substrate analogues indicates that the phosphate moiety of the substrate is required for binding and that substrate analogues in the open chain form bind more effectively to the enzyme than the corresponding cyclic compounds. A reaction mechanism is proposed in which the zinc coordinates with the carbonyl and hydroxyl oxygens on C-1 and C-2, thereby activating the hydrogen α to the carbonyl, which is abstracted by the nonprotonated nitrogen of an imidazole group to form the transient enediol intermediate.