A systematic analysis of acceptor specificity and reaction kinetics of five human α(2,3)sialyltransferases: Product inhibition studies illustrate reaction mechanism for ST3Gal-I

Abstract

Sialyltransferases (STs) catalyze the addition of sialic acids to the non-reducing ends of glycoproteins and glycolipids. In this work, we examined the acceptor specificity of five human α(2,3)sialyltransferases, namely ST3Gal-I, -II, -III, -IV and -VI. KM values for each of these enzymes is presented using radioactivity for acceptors containing Type-I (Galβ1,3GlcNAc), Type-II (Galβ1,4GlcNAc), Type-III (Galβ1,3GalNAc) and Core-2 (Galβ1,3(GlcNAcβ1,6)GalNAc) reactive groups. Several variants of acceptors inhibited ST3Gal activity emphasizing structural role of acceptor in enzyme-catalyzed reactions. In some cases, mass spectrometry was performed for structural verification. The results demonstrate human ST3Gal-I catalysis towards Type-III and Core-2 acceptors with KM = 5–50μM and high VMax values. The KM for ST3Gal-I and ST3Gal-II was 100 and 30-fold lower, respectively, for Type-III compared to Type-I acceptors. Variants of Type-I and Type-II structures characterized ST3Gal-III, -IV and -VI for their catalytic specificity. This manuscript also estimates KM for human ST3Gal-VI using Type-I and Type-II substrates. Together, these findings built a platform for designing inhibitors of STs having therapeutic potential.

Structural basis for mammalian nucleotide sugar transport

“…This concentration gradient of NMPs across the Golgi membrane accounts for the observation that CST, like many other NSTs, is a secondary active transporter and can concentrate nucleotide sugars inside the Golgi lumen (Hirschberg et al, 1998). This is an important property of this system as many glycosyltransferases have K m ’s for nucleotide sugars that can approach mM concentrations and sialyltransferase K m ’s in particular range from 0.05 to 3.2 mM (Gupta et al, 2016). The kinetics of substrate efflux from the Golgi through CST are not well defined; however, high lumenal CMP concentrations coupled with CMP’s relatively high binding affinity for the partially-occluded state will ensure efficient conversion of CST back to a cytoplasmic-facing state.…”

A Systematic Review on the Implications of O-linked Glycan Branching and Truncating Enzymes on Cancer Progression and Metastasis

“…These enzymes are mostly specific to the endoplasmic reticulum (ER), Golgi, and the extracellular milieu [2][3][4]. Reactions that are catalyzed by glycosyltransferases occur through a bi-bi substrate mechanism, wherein a sugar-nucleotide donor and a carbohydrate acceptor participate to form a modified glycan and a nucleoside as the products [5]. The complexity in glycosylation occurs due to the inherent stochasticity arising from the enzyme concentration, transport phenomenon, kinetic parameters, thermodynamics, and sugar-nucleotide transporters [6,7].…”

A mechanism for bistability in glycosylation

“…Whereas a rapid-equilibrium random-order mechanism is a feature of polypeptide N -acetylgalactosaminyltransferase [ 20 ], sulfotransferases [ 21 ], fucosyltransferases [ 22 ] and sialyltransferases [ 23 ], with other glycosyltransferases, such as those of the N -acetylglucosaminyltransferase and galactosyltransferase families, the enzyme must bind the donor first, before catalysis can occur [ 24 ]. Under quasi-steady-state conditions ( Fig 1B ), the rate law for the compulsory order binding is (Eq S3 in S1 Appendix ): In such a case, the inhibitory effect of multi-substrate competition will lessen as the concentration of the donor is increased towards saturating levels.…”