The adenine nucleotide binding site on yeast hexokinase PII. Affinity labeling of Lys-111 by pyridoxal 5'-diphospho-5'-adenosine.
The adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), is shown to be a potent and specific inhibitor of yeast hexokinase PII. Evidence that the analog binds specifically at the ATP binding site includes the demonstration that glucose binding enhances PLP-AMP binding and that PLP-AMP and ATP bind competitively with an apparent Ki(PLP-AMP) = 23 microM. In addition, from the relationship between the degree of inhibition and extent of modification, it is estimated that the incorporation of 1 mol of PLP-AMP/mol of subunit is required for complete inhibition. Borohydride reduction of the Schiff's base complex formed between hexokinase and [3H]PLP-AMP gives a stable product. The reduced derivative was digested with trypsin and a single radioactive peptide was isolated by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis identified Lys-111 as the modified residue. Taking into account the known structures of the binary complexes (Shoham, M., and Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14), the results suggest that Lys-111, located in the smaller of the two lobes of hexokinase, moves into the active site upon formation of the ternary complex.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D001667:Binding, Competitive; D003198:Computer Simulation; D006593:Hexokinase / Q000037:antagonists & inhibitors*; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D000227:Adenine Nucleotides; D000345:Affinity Labels; D007527:Isoenzymes; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine