Use of Synthetic Oligosaccharide Substrate Analogs to Map the Active Sites of N-Acetylglucosaminyltransferases I and II

Publisher Summary This chapter focuses on the use of synthetic oligosaccharide substrate analogs to map the active sites of N -acetylglucosaminyltransferases I and II. The synthesis of complex N-glycans can be divided into three distinct stages: the first stage occurs primarily in the cytoplasm and rough endoplasmic reticulum; the second stage begins with the transfer of Glc3Man9GlcNAc; the third stage occurs primarily in the Golgi apparatus. Catalysis by both GlcNAc-T I and II occurs by an ordered sequential Bi–Bi mechanism in which UDP-GlcNAc/Mn2+ binds first and UDP leaves last. The substrate analog-binding data described in the chapter were obtained with partially purified preparations of full-length nonrecombinant bovine colostrums and rat liver GlcNAc-T I and full-length nonrecombinant rat liver GlcNAc-T II. The chapter also discusses the binding of substrate analogs to N -acetylglucosaminyltransferase I, the binding of substrate analogs to N -acetylglucosaminyltransferase II, and the correlation of substrate –analog-binding data with the X-Ray crystal structure of N -acetylglucosaminyltransferase I.