Reaction mechanism and specificity of human GMP reductase. Substrates, inhibitors, activators, and inactivators.

GMP reductase was purified 150,000-fold from human erythrocytes by affinity chromatography on Cibacron blue/agarose. The enzyme was stable to storage and could be assayed spectrophotometrically. Plots of the velocity-* uersus [GMP]-’ demonstrated downward curvature above 250 pM GMP. GTP was found to be a nonessential activator. It caused the K’, for GMP to decrease from 7.5 to 4.4 pM and the V& to increase by 40%. At 21 pM GMP, saturating GTP increased the reaction velocity by 44%. Half-maximal increase occurred at 2.0 yM GTP. Micromolar and submicromolar concentrations of GDP, 2’dGTP, 2’-dGDP, and Gp,G also activated the enzyme. The downward curvature of the GMP-saturation plot was probably due to the binding of GMP to the activator site. The enzyme was found to have a narrow substrate specificity. Nine analogs of GMP were examined for alternative substrate activity. Six of these could bind competitively with respect to GMP, but only three were substrates. These substrates (K’,), araGMP (190 FM), 2’dGMP (8.2 PM), and 8-azaGMP (4.2 PM), had V& values only 1 to 4% of that of GMP and therefore appear to be potential alternate-substrate inhibitors. A study of four analogs of NADPH revealed that the enzyme markedly prefers NADPH as the coenzyme. Xanthosine monophosphate (XMP) was a competitive inhibitor with respect to GMP. The Ki,, 0.17 pM, was found to be equal to the dissociation constant of XMP from the enzyme*XMP complex. XMP was a noncompetitive inhibitor with respect to NADPH. When XMP and GTP were simultaneously present, there were no changes in the dissociation constant of XMP or the activatedV&,,. Therefore, the activation and inhibition appeared to be independent processes. Five analogs of XMP were potent competitive inhibitors with respect to GMP. These inhibitors and their dissociation constants are: &azaXMP, 0.3 PM; 6thioXMP, 2.0 pM; araXMP, 2.5 pM; 8-aza-7-deazaXMP, 5.0 PM; 2’-dxMP, 27 PM. 6-ThioGMP, 6-thioIMP, and 6-chloropurine ribonucleotide caused apparent inactivation of the enzyme. The concentration of inactivator required to produce inactivation varied with different batches of enzyme. Inactivation could be either prevented or reversed by exogenous sulfhydryl-containing compounds. GMP, but not NADPH, could retard the rate at which the inactivation occurred. 6-ThioXMP did not inactivate the enzyme. The kinetic analysis and the inhibition patterns of XMP and two other inhibitors are most consistent with