Benjamin A. Horenstein

Synthesis of tunichromes mm-l and mm-2, blood pigments of the iron.Assimilating tunicate, molgula manhattensis

Abstract Unstable poly-phenolic pigments, tunichromes Mm-1 and Mm-2, blood pigments of the iron-assimilating tunicate, Molgula manhattensis, have been synthesized by a modified versatile route.

Primary 13C and beta-secondary 2H KIEs for trans-sialidase. A snapshot of nucleophilic participation during catalysis.

Trypanosoma cruzi trans-sialidase catalyzes a novel reaction that involves the transfer of sialic acid between host and parasite glycoconjugates. In this paper, we report kinetic isotope effect studies on recombinant trans-sialidase. beta-Dideuterium and primary 13C isotope effects were measured for a good substrate, sialyl-lactose, and a slow substrate, sialyl-galactose, in both acid-catalyzed solvolysis and enzymatic transfer reactions. The beta-dideuterium isotope effect for sialyl-lactose in the acid hydrolysis reaction was 1.113 +/- 0.012. The primary 13C isotope effects for hydrolysis of sialyl-lactose and sialyl-galactose were 1. 016 +/- 0.011 and 1.015 +/- 0.008, respectively. In the enzymatic transfer reactions, the beta-dideuterium and primary 13C effects for sialyl-galactose were 1.060 +/- 0.008 and 1.032 +/- 0.008, respectively. The isotope effects for hydrolysis describe a dissociative SN1-like mechanism, and these data are contrasted by the data for the enzyme-catalyzed reaction. The enzymatic deuterium isotope effects are lower by a factor of 2, but the primary carbon isotope effects are higher by a factor of 2. This pattern describes a mechanism involving nucleophilic participation in the rate-determining transition state.

Characterization of alpha(2-->6)-sialyltransferase reaction intermediates: use of alternative substrates to unmask kinetic isotope effects.

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.