Characterization of a (2 → 6)-sialyltransferase reaction intermediates: Use of alternative substrates to unmask kinetic isotope effects

Abstract

This chapter illustrates the way in which the rival explanations can be addressed by employing a poorer alternative substrate to unmask intrinsic kinetic isotope effects and to reveal significant mechanistic details about sialyltransferase catalysis. Enzyme action on a preferred substrate is often characterized by rate processes that lead to high catalytic efficiency but also prevent a thorough kinetic analysis of the reaction mechanism. This limitation frequently can be obviated by the use of alternative substrates that exhibit lower rates of catalysis and often unmask kinetic features that are indiscernible with the natural substrate. In the case of α(2→6)- sialyltransferase, the alternative donor substrate uridine monophosphate glycoside of N-acetylneuraminic acid (UMP-NeuAc) provides the opportunity to probe the mechanism in greater detail than achieved with cytidine monophosphate glycoside of N-acetylneuraminic acid (CMP-NeuAc). The specificity of CMP-NeuAc synthase for cytidine triphosphate (CTP) posed a synthetic challenge for the preparation of UMP-NeuAc isotopomers by direct enzymatic means from uridine triphosphate (UTP) and labeled N-acetylneuraminic acid (NeuAc). This was overcome by use of diazotization and subsequent hydrolysis to convert radioisotopically labeled CMP-NeuAc isotopomers directly to the corresponding UMP-NeuAc isotopomers, thereby greatly facilitating analysis of kinetic isotope effects.