Transient kinetic and deuterium isotope effect studies on the catalytic mechanism of phosphoglycerate dehydrogenase.
The catalytic mechanism of the phosphoglycerate dehydrogenase reaction in both directions was investigated by studying: (a) pre-steady state transients in reduced coenzyme appearance or disappearance or disappearance and in protein fluorescence; (b) deuterium isotope effects on the transients and on the steady state reactions; and (c) the partial reaction between the enzyme-NADH complex and hydroxypyruvate-P. These studies led to the scheme below for the ternary complex interconversion. E1-NADH-hydroxypyruvate-P(1)equilibriumE2-NADH-hydroxypyruvate-P(2)equilibriumE3-NADH-hydroxypyruvate-P + H+(3)equilibriumE3-NAD+-3-phosphoglycerate(4)equilibriumE4-NAD+-3-phosphoglycerate Steps 1,2, and 4 are ternary complex isomerizations. Step 3 is the hydride transfer. Under steady state conditions isomerization 2 is the rate-determining step in the direction of hydroxypyruvate-P reduction at higher pH values. At lower pH values, the hydride transfer step is also partially rate-determining. The rate-determining step in the direction of 3-phosphoglycerate oxidation occurs subsequent to the hydride transfer step at higher pH values. At lower pH values the rate is determined by both isomerization 4 and the hydride transfer step. Isomerizations 1, 2, and 4 were inhibited by serine, an allosteric inhibitor, indicating that the inactive conformation of the enzyme is incapable of performing any of the steps of the ternary complex interconversion. Phosphoglycerate dehydrogenase corresponds to a V-type allosteric enzyme. When the enzyme-NADH complex was mixed with hydroxypyruvate-P at pH 8.5, a rapid quenching of enzymebound NADH fluorescence occurred. This process was studied under pseudo-first order conditions and shown to be the result of hydroxypyruvate-P binding.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D003201:Computers; D003903:Deuterium / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D012694:Serine / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D012694:Serine; D003903:Deuterium; D000429:Alcohol Oxidoreductases; D002237:Carbohydrate Dehydrogenases