Yeast hexokinase PII--bound nucleotide conformation at the active site.

摘要: The structure of adenine nucleotide bound at the active site yeast hexokinase PII (PII) was studied in complexes · ADPMg(II), ADPMg(II) Glc and NO3−· using two-dimensional transferred NOE spectroscopy. Binding ligand to enzyme resulted downfield shift several resonances. Changes chemical as a function concentration indicate that various resonances free form appear be fast exchange. largest change between states (ΔvM= 88 ± 9 Hz) an NMR frequency 500 MHz observed for H2 resonance ring. A lower limit rate dissociation from complex derived these results is koff > 550 −1 Interproton NOEs protons NO−3· were measured 10°C. buildup curves constructed such measurements made mixing times (40, 80, 120, 160 200 ms) analyzed complete relaxation matrix calculations interproton distances determined. These used restrained molecular dynamics derive conformation enzyme. binds anti conformation. glycosidic torsion angle x(O4′-C1′-N9-C8) determined are 68 4o, 52 4o 49 respectively. In all complexes, ribose pucker best represented by unsymmetrical O4′-endo-C1′-exo twist (oT1). decrease attributed glucose-induced conformational changes here variance one proposed on basis X-ray crystallography model building [Shoham, M. & Steitz, T. A. (1980) J. Mol. Biol. 140, 1–14].