Circular dichroic spectroscopy of N-acetylglucosaminyltransferase V and its substrate interactions.
beta-1,6-N-Acetylglucosaminyltransferase V (EC 2.4.1.155) catalyzes the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc in beta(1,6)-linkage to the alpha(1,6)-linked mannose of N-linked oligosaccharides. Circular dichroism (CD) was used to investigate the secondary structure 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-structure and substantial amounts (>50%) of alpha-helicity. The CD spectra of solutions containing UDP-GlcNAc and different ratios of UDP-GlcNAc:enzyme were measured. Interestingly, the spectrum of each mixture could not be accounted for by simple additivity of the two individual spectra, indicating a change in environment of the chromophores and/or a conformational change of the substrate or protein concomitant with binding. Similar results were obtained with mixtures of UDP and the enzyme. Analysis of the CD difference spectra at three wavelengths yielded an estimated average Kd of 4.4 mM for UDP-GlcNAc and 3.8 mM for UDP. By contrast, addition of the CD spectrum of an inhibitory substrate analog 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 enzyme did not alter the Kd associated with UDP-GlcNAc binding to the enzyme. These results and kinetic studies reported herein suggest an ordered reaction in which UDP-GlcNAc binds first to the enzyme, followed by the sequential binding of the trisaccharide substrate.
Zhang
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Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Peng
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KC
Chen
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Puett
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D
Pierce
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M
D002240:Carbohydrate Sequence; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase