RESUMO
The synthesis of UDP-N-trifluoroacetylglucosamine [uridine 5'-(2-trifluoroacetamido-2-deoxy-alpha-D-glucopyranosyl diphosphate, UDP-GlcNAc-F3] is reported. The compound is found to serve as a substrate for the core-2 GlcNAc transferase (EC 2.4.1.102) that is involved in the biosynthesis of O-linked glycoproteins and for the GlcNAcT-V transferase (EC 2.4.1.155) that is a key biosynthetic enzyme controlling the branching pattern of cell surface complex Asn-linked oligosaccharides. The trisaccharide beta-D-Galp-(1-->3) -[beta-D-GlcpNAc-F3(1-->6)] alpha-D-GalpNAc-OR [R = (CH2)8CO2Me] was prepared from beta-D-Galp-(1-->3) -alpha-D- GalpNAc-OR using the core-2 GlcNAc transferase. The tetrasaccharide beta-D-GlcpNAc-(1 -->2)-[beta-D-GlcpNAc-F3-(1-->6)]-alpha-D-Manp-(1-->6)-beta-D-Glcp -OR [R = (CH2)7CH3] using the GlcNAcT-V transferase. Removal of the trifluoroacetyl group was achieved under mild basic conditions to give the corresponding glucosamine containing tetrasaccharide. These examples demonstrate the feasibility of introducing masked forms of glucosamine residues into oligosaccharides using GlcNAc-specific transferases. The requirement for the trifluoroacetamido group as a specific recognition element was evident in the observation that neither UDP-glucosamine nor UDP-glucose served as a donor substrates for the core-2 GlcNAc transferase.
Assuntos
Acetilglucosamina/análogos & derivados , N-Acetilglucosaminiltransferases/metabolismo , Difosfato de Uridina/análogos & derivados , Acetilglucosamina/síntese química , Acetilglucosamina/metabolismo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Estudos de Viabilidade , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Rotação Ocular , Ratos , Relação Estrutura-Atividade , Especificidade por Substrato , Temperatura , Difosfato de Uridina/síntese química , Difosfato de Uridina/metabolismoRESUMO
UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7 microM) of the enzyme. The enzyme is irreversibly inactivated by uridine 5'-diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (ki/Ki) was found to be 2 x 10(3) mM-1 min-1. Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5'-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.
