pH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 1. Alternate substrates oxalacetate, glycolate, hydroxylamine, and fluoride.
The decarboxylation of oxalacetate shows equilibrium-ordered kinetics, with Mg2+ adding before oxalacetate. The Ki for Mg2+ increases below a pK of 6.9, corresponding to a ligand of the metal that is probably glutamate, and decreases above a pK of 9.2, corresponding to water coordinated to enzyme-bound Mg2+. Both V and V/KOAA decrease above the pK of 9.2, suggesting that the carbonyl oxygen of oxalacetate must replace water in the inner coordination sphere of Mg2+ prior to decarboxylation. The enzyme-Mg2+-oxalacetate complex must be largely an outer sphere one, however, since the pK of 9.2 is seen in the V profile. The phosphorylation of glycolate or N-hydroxycarbamate (the actual substrate that results from reaction of hydroxylamine with bicarbonate) occurs only above the pK of 9.2, with V/K profiles decreasing below this pH. The alkoxides of these substrates appear to be the active species, replacing water in the coordination sphere of Mg2+ prior to phosphorylation by MgATP. Glycolate, but not N-hydroxycarbamate, can bind when not an alkoxide, since the V profile for the former decreases below a pK of 8.9, while V for the latter is pH independent. Initial velocity patterns for phosphorylation of fluoride in the presence of bicarbonate show saturation by MgATP but not by fluoride. The V/K profile for fluoride decreases above the pK of 9.0, showing that fluoride must replace water in the coordination sphere of Mg2+ prior to phosphorylation. None of the above reactions is sensitive to the protonation state of the acid-base catalyst that assists the enolization of pyruvate in the physiological reaction.
D000818:Animals; D005459:Fluorides / Q000378:metabolism*; D006016:Glycolates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D010071:Oxaloacetates / Q000378:metabolism*; D010766:Phosphorylation; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits
D006016:Glycolates; D006898:Hydroxylamines; D010071:Oxaloacetates; C031149:glycolic acid; D019811:Hydroxylamine; D011770:Pyruvate Kinase; D005459:Fluorides