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

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 alpha(2,3)sialyltransferases, namely ST3Gal-I, -II, -III, -IV and -VI. K-M values for each of these enzymes is presented using radioactivity for acceptors containing Type-I (Gal beta 1,3GlcNAc), Type-II (Gal beta 1,4GIcNAc), Type-III (Gal beta 1,3GalNAc) and Core-2 (Gal beta 1,3(GlcNAc beta 1,6)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 K-M = 5-50 mu M and high V-Max values. The K-M for ST3Gal-I and ST3Ga1-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 K-M 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. (C) 2015 Elsevier Inc. All rights reserved.