Critical ionization states in the reaction catalyzed by triosephosphate isomerase.
To allow the detailed interpretation of the pH dependences of the steady-state parameters for the reaction catalyzed by triosephosphate isomerase, three kinds of experiments have been performed. First, the value of kcat/Km for enzyme-catalyzed isomerization of the phosphonate analogue of D-glyceraldehyde 3-phosphate (2-hydroxy-4-phosphonobutyraldehyde) has been shown to titrate with an apparent pKa of 7.5, which is close to the phosphonate's second ionization constant. Secondly, the sulfate ester analogue of dihydroxyacetone phosphate (dihydroxyacetone sulfate), which exists only as a monoanion over the pH range of interest, has been shown not to bind detectably to the enzyme. Thirdly, an isotopic discrimination experiment at pH 5.2 has been compared with a similar investigation at pH 7.6. The results together demonstrate that both enzyme and substrate ionizations control the reaction rate in the pH range 5 to 8.
Critical ionization states in the reaction catalyzed by triosephosphate isomerase. To allow the detailed interpretation of the pH dependences of the steady-state parameters for the reaction catalyzed by triosephosphate isomerase, three kinds of experiments have been performed. First, the value of kcat/Km for enzyme-catalyzed isomerization of the phosphonate analogue of D-glyceraldehyde 3-phosphate (2-hydroxy-4-phosphonobutyraldehyde) has been shown to titrate with an apparent pKa of 7.5, which is close to the phosphonate's second ionization constant. Secondly, the sulfate ester analogue of dihydroxyacetone phosphate (dihydroxyacetone sulfate), which exists only as a monoanion over the pH range of interest, has been shown not to bind detectably to the enzyme. Thirdly, an isotopic discrimination experiment at pH 5.2 has been compared with a similar investigation at pH 7.6. The results together demonstrate that both enzyme and substrate ionizations control the reaction rate in the pH range 5 to 8.