title: 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 = 5-50 μM 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.
authors.affiliation: Department of Chemical and Biological Engineering, State University of New York, Buffalo, NY 14260, USA. Electronic address: rohitesh.gupta@gmail.com.
authors.forename: Rohitesh
authors.initials: R
authors.lastname: Gupta
authors.affiliation: Tumor End LLC, Baton Rouge, LA 70803, USA.
authors.forename: Khushi L
authors.initials: KL
authors.lastname: Matta
authors.affiliation: Department of Chemical and Biological Engineering, State University of New York, Buffalo, NY 14260, USA; NY State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY 14260, USA. Electronic address: neel@buffalo.edu.
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D006017:Glycolipids / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D012794:Sialic Acids / Q000737:chemistry*; D012799:Sialyltransferases / Q000737:chemistry*; D013379:Substrate Specificity; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D006017:Glycolipids; D006023:Glycoproteins; D012794:Sialic Acids; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase; C000722087:ST3GAL1 protein, human
keywords: Mass spectrometry; Michaelis–Menten; Product inhibition; Sialyltransferase