CIRCULAR DICHROIC SPECTROSCOPY OF N-ACETYLGLUCOSAMINYLTRANSFERASE-V AND ITS SUBSTRATE INTERACTIONS

beta-1,6-N-Acetylglucosaminyltransferase V (EC 2.4.1.155) catalyzes th e transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc in beta(1,6 )-linkage to the alpha(1,6)-linked mannose of N-linked oligosaccharide s. Circular dichroism (CD) was used to investigate the secondary struc ture of a recombinant, soluble form of the enzyme and its interaction with UDP-GlcNAc and an inhibitory substrate analog. The CD spectrum of the apoenzyme indicated the presence of small amounts of beta-structu re and substantial amounts (>50%) of alpha-helicity. The CD spectra of solutions containing UDP-GlcNAc and different ratios of UDP-GlcNAc:en zyme were measured. Interestingly, the spectrum of each mixture could not be accounted for by simple additivity of the two individual spectr a, indicating a change in environment of the chromophores and/or a con formational change of the substrate or protein concomitant with bindin g. Similar results were obtained with mixtures of UDP and the enzyme, Analysis of the CD difference spectra at three wavelengths yielded an estimated average K-d of 4.4 mM for UDP-GlcNAc and 3.8 mM for UDP. By contrast, addition of the CD spectrum of an inhibitory substrate analo g of its oligosaccharide acceptor substrate and the CD spectrum of the enzyme could account for that observed of an inhibitor-enzyme mixture ; moreover, addition of the inhibitor to a mixture of UDP-GlcNAc and e nzyme did not alter the K-d associated with UDP-GlcNAc binding to the enzyme. These results and kinetic studies reported herein suggest an o rdered reaction in which UDP-GlcNAc binds first to the enzyme, followe d by the sequential binding of the trisaccharide substrate.