Presence of an essential lysine residue in a GDP-fucose protected site of the alpha 1----3fucosyltransferase from human small cell lung carcinoma NCl-H69 cells.
The NCI-H69 cell alpha 1----3fucosyltransferase has been purified from a 0.2% Triton X-100R solubilized enzyme fraction by GDP-hexanolamine-Sepharose affinity chromatography and Superose 12 gel filtration. Photoaffinity labeling experiments with 125I-GDP-hexanolaminyl-4-azidosalicylic acid present in concentrations equivalent to 0.5 and 1 times Ki of the inhibitor for the enzyme indicated that labeling of the 45-kDa protein band could be inhibited by addition of 400 microM GDP-fucose but was not effected by similar concentrations of either GDP-mannose or GDP-glucose. The purified enzyme was applied to studies intended to define catalytically essential amino acid residues of the protein. Incubation of the enzyme in the presence of increasing concentrations of pyridoxal 5'-phosphate was found to result in irreversible inactivation of the enzyme after NaBH4 reduction. The donor substrate, GDP-fucose, was found to protect the enzyme from inactivation. Little or no protection was found for either GDP-mannose or the acceptor substrate nLc4. Pyridoxal 5'-phosphate was shown to behave as a competitive inhibitor with respect to GDP-fucose with a Ki of 105 microM. Labeling with 3H-pyridoxal 5'-phosphate resulted in the incorporation of approximately 8 mol pyridoxal 5'-phosphate per mole subunit. Parallel experiments containing GDP-fucose indicated protection of one site per subunit correlated with GDP-fucose binding. Acid hydrolysis and chromatographic analysis of the 3H-pyridoxylated protein indicated greater than 95% of the 3H label was recovered as pyridoxyl-lysine irrespective of whether GDP-fucose was present or not during labeling. These studies indicate the presence of a catalytically essential lysine residue associated with GDP-fucose binding to this enzyme. This information will be of value in further studies of this and other alpha 1----3fucosyltransferases and may suggest a practical basis for modulation of enzyme activity in the cell.
Holmes
E H
EH
Pacific Northwest Research Foundation, Seattle, Washington 98122.
D001665:Binding Sites; D001667:Binding, Competitive; D001894:Borohydrides / Q000494:pharmacology; D018288:Carcinoma, Small Cell / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology*; D008239:Lysine / Q000737:chemistry*; D010084:Oxidation-Reduction; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D001894:Borohydrides; D006154:Guanosine Diphosphate Fucose; D011732:Pyridoxal Phosphate; C025364:sodium borohydride; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D008239:Lysine