Solvent Deuterium Isotope Effects on the Glyceraldehyde 3-Phosphate Dehydrogenase-catalyzed Hydrolysis of p-Nitrophenyl Acetate
Robert N. Lindquist 1 and Eugene H. Cordes 1
From the
1 From the Department of Chemistry, Indiana University, Bloomington, Indiana 47401
Solvent deuterium isotope effects on the kinetics of glyceraldehyde 3-phosphate dehydrogenase-catalyzed hydrolysis of p-nitrophenyl acetate in aqueous solution in the pH range 6 to 9 and at 25° have been investigated. With the use of enzymes isolated both from rabbit muscle and yeast, it has been established that the rate of enzyme acylation by this ester is decreased 2.5-fold in deuterium oxide compared to water under conditions in which the rate is linearly dependent on hydroxide ion concentration but is independent of the isotopic nature of the solvent under more basic conditions in which the rate is independent of the hydroxide ion concentration. In water, the acylation rate constant is dependent on a basic group, the conjugate acid of which has a pKa near 8.2; in deuterium oxide this same group has a pKa near 8.7. This behavior is consistent with an acylation reaction involving the unaided attack of the anionic form of a crucial sulfhydryl residue on p-nitrophenyl acetate within the Michaelis-Menten complex. Closely related behavior is observed for deacylation of the yeast enzyme: the rate is depressed 3-fold by deuterium oxide under conditions of hydroxide ion catalysis but is unaffected by this agent under more basic conditions in which the reaction is pH-independent. Values of pKa for the conjugate acid of the basic species upon which the reaction depends are 8.8 and 9.3 in water and deuterium oxide, respectively.
Submitted on June 17, 1968
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