Active site water molecules revealed in the 2.1 angstrom resolution structure of a site-directed mutant of isocitrate dehydrogenase

Isocitrate dehydrogenase catalyses the two step, acid base, oxidative decar boxylation of isocitrate to alpha-ketoglutarate, Lysine 230 was suggested t o act as proton donor based on geometry and spatial proximity to isocitrate . To clarify further the role of lysine 230, we co-crystallized the lysine- to-methionine mutant (K230M) with isocitrate and with alpha-ketoglutarate. Crystals were flash-frozen and the two structures were determined and refin ed to 2.1 Angstrom. Several new features were identified relative to the wi ld-type structure. Seven side-chains previously unplaced in the wildtype st ructure were identified and included in the model, and the amino acid termi nus was extended by an alanine residue. Many additional water molecules wer e identified. Examination of the K230M active sites (K230M isocitrate and K230M-ketogluta rate) revealed that tyrosine 160 protrudes further into the active site in the presence of either isocitrate or alpha-ketoglutarate in K230 M than it does in the wild-type structure. Also, methionine 230 was not as fully exte nded, and asparagine 232 rotates similar to 30 degrees toward the ligand pe rmitting polar interactions. Outside the active site cleft a tetragonal vol ume of density was identified as a sulfate molecule. Its location and inter actions suggest it may influence the equilibrium between the tetragonal and the orthorhombic forms of isocitrate dehydrogenase. Differences observed i n the active site water structure between the wild-type and K230M structure s were due to a single point mutation. A water molecule was located in the position equivalent to that occupied by the wild-type epsilon-amine of lysi ne 230; a water molecule in that location in K230M suggests it may influenc e catalysis in the mutant. Comparison of K230M complexed with isocitrate an d alpha-ketoglutarate illuminates the influence a ligand has on active site water structure. (C) 2000 Academic Press.