RESUMO
Two detailed mechanisms [Marks et al. (2001) Biochemistry 40, 6805] have been proposed to explain the activity of methylglyoxal synthase (MGS), a homohexameric allosterically regulated enzyme that catalyzes the elimination of phosphate from DHAP to form enol pyruvaldehyde. This enol then tautomerizes to methylglyoxal in solution. In one of these mechanisms His 98 plays an obligate role in the transfer of a proton from the O(3) oxygen of DHAP to the O2 oxygen. To test this hypothesized mechanism, the variants H98N and H98Q were expressed and purified. Relative to the wild-type enzyme, the H98N variant shows a 50-fold decrease in k(cat) with all other kinetic parameters (i.e., K(m), K(PGA), etc.) being nearly the same. By contrast, the apparent catalytic rate for the H98Q variant is 250-fold lower than that of the wild-type enzyme. Inorganic phosphate acts as a competitive inhibitor (with a K(i) of 15 microM) rather than as an allosteric-type inhibitor as it does in the wild-type enzyme, and the competitive inhibitor phosphoglyolate (PGA) acts as an activator of this variant. These facts are explained by a shift in the conformational equilibrium toward an "inactive" state. When activation by PGA is accounted for, the catalytic rate for the "active" state of H98Q is estimated to be only 6-fold less than that of the wild-type enzyme, and thus His 98 is not essential for catalysis. Primary deuterium isotope effect data were measured for the wild-type enzyme and the two variants. At pH 7.0, the (D)V isotope effect (1.5) and the absence of a (D)(V/K) isotope effect for the wild-type enzyme suggest that the rate for the isotopically sensitive step is partially rate limiting but greater than the rate of substrate dissociation. Both the (D)V (2.0) and (D)(V/K) (3.4) isotope effects were more pronounced in the H98N variant, while the H98Q variant displayed an unusual inverse (D)V (0.8) isotope effect and a normal (D)(V/K) (1.5) isotope effect. The X-ray crystal structures of PGA bound to the H98Q and H98N variants were both determined to a resolution of 2.2 A. These mutations had little effect on the overall structure. However, the pattern of hydrogen bonding in the active site suggests an explanation as to how in the wild-type and H98N mutated enzymes the "inactive to active" equilibrium lies toward the active state, while with the H98Q mutated enzyme the equilibrium lies toward the inactive state. Thus, the role of His 98 appears to be more as a regulator of the enzyme's conformation rather than as a critical contributor to the catalytic mechanism.
