J. Biol. Chem., Vol. 263, Issue 16, 7907-7912, Jun, 1988
The adenine nucleotide binding site on yeast hexokinase PII. Affinity labeling of Lys-111 by pyridoxal 5'-diphospho-5'-adenosine
JK Tamura, JR LaDine and RL Cross
Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210.
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.
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