Primary¹³C and β-Secondary²H KIEs for Trans-sialidase. A Snapshot of Nucleophilic Participation during Catalysis

Abstract

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.

The Antimicrobial Defense of the Pacific Oyster, Crassostrea gigas. How Diversity may Compensate for Scarcity in the Regulation of Resident/Pathogenic Microflora

“…They contain from six to eight cysteine residues. This motif is widespread in fungi and in invertebrate defensins, like in arthropods and mollusks (Yang et al, 2000; Bulet and Stöcklin, 2005; Mygind et al, 2005), but the presence of eight cysteine residues was only reported in species of bivalve mollusks (Hubert et al, 1996; Gueguen et al, 2006; Gonzalez et al, 2007a). CSαβ-containing defensins have no evident phylogenetic relationships with the vertebrate defensins and big defensins (Rosa et al, 2011).…”

Use of conformationally restricted pyridinium α-D-N-acetylneuraminides to probe specificity in bacterial and viral sialidases

“…That is, the conserved tyrosine residue acts as a nucleophile, presumably with general-base catalyzed assistance from the conserved glutamate. Also, based on a kinetic isotope effect study, nucleophilic attack is concerted with leaving-group departure for the Trypanosoma cruzi transsialidase catalyzed reaction of 2,3-α-D-sialyl-galactose (Yang et al 2000). The mechanism displayed in Scheme 1 is a refinement of several previous proposals (Chong et al 1992;Varghese and Colman 1991;Varghese et al 1992), but is inconsistent, at least in the case of trans-sialidases (Yang et al 2000), with the formation of a discrete cationic intermediate (Taylor and von Itzstein 1994;Thomas et al 1999).…”

“…Also, based on a kinetic isotope effect study, nucleophilic attack is concerted with leaving-group departure for the Trypanosoma cruzi transsialidase catalyzed reaction of 2,3-α-D-sialyl-galactose (Yang et al 2000). The mechanism displayed in Scheme 1 is a refinement of several previous proposals (Chong et al 1992;Varghese and Colman 1991;Varghese et al 1992), but is inconsistent, at least in the case of trans-sialidases (Yang et al 2000), with the formation of a discrete cationic intermediate (Taylor and von Itzstein 1994;Thomas et al 1999). The roles of other active-site residues are as follows: the conserved aspartic acid acts as a general-acid catalyst (Watson et al 2004); and the arginine triad mediates strong electrostatic binding to the anionic carboxylate of the N-acetylneuraminide substrate (Vimr 1994).…”

Chemoenzymatic synthesis of ring 18O-labeled sialic acid

“…Specifically, kinetic isotope effects (KIEs) measured using compounds derived from (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) O)-sialic acid (1b), in conjunction those from anomeric 13 C-and leaving group 18 Olabeled materials, will allow evaluation of critical variables at the transition state(s) for the transfer of sialic acid residues, namely: (i) site of cleavage at the glycosidic centre, endo-or exo-cyclic; (ii) degree of C-O cleavage; (iii) amount of double-bond character at the oxacarbenium ion-like TS; (iv) degree of nucleophilic participation at the anomeric centre; (v) degree of proton-transfer, which occurs from an active-site aspartic acid residue for the enzymecatalyzed reactions (7,8).…”

“…Those include leaving-group 18 O-KIEs on the acid-catalyzed and spontaneous hydrolysis reactions of 4-nitrophenyl α-D-sialoside (9) and on the corresponding Vibrio cholerae sialidase-catalyzed reactions (10); anomeric 14 C-KIEs on the sialyl transferase catalyzed transfer of sialic acid from CMP-and UMP-β-sialoside (2 and 3, respectively; Fig. 1) to carbohydrate acceptors (11,12); anomeric 14 C-KIEs on the hydrolysis of sialosides catalyzed by the trans-sialidase Trypanosoma cruzi (13).…”