Synthesis and properties of 5-azido-UDP-glucose: Development of photoaffinity probes for nucleotide diphosphate sugar binding sites

Abstract

Abstract A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.