HIGH RESOLUTION STRUCTURE OF HUMAN ORNITHINE AMINOTRANSFERASE (OAT) COMPLEXED WITH alpha-KETOGLUTARAT: IMPLICATIONS OF THE REACTION MECHANISM. Betty W. Shen, Sapan A. Shah, and Axel T. Brunger. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA

The crystal structure of an OAT/alpha-ketoglutarate complex is refined against a 2.0Å resolution data set collected at -150deg.C using a CCD dector at CHESS with synchrotron radiation of 0.92Å wavelength. The data is 99.6% complete with Rsymm = 7.2% (25.4%). The unit cell dimensions of the frozen crystal are slightly smaller than the data set collected at ambient temperature, which was used for solving the structure of OAT (Shen et al., manuscript in preparation). The refined coordinates at 2.5Å resolution was used directly for the refinement of the enzyme-substrate complex. A strong, continuous tube of electron density in the partially refined density map of all three protomers in the asymmetric unit suggested the presence of a molecule of alpha-ketoglutarate in the active site pocket.The presence of the substrate molecules is further confirmed by its persistence in a simulated-annealing omit map.

Interestingly, the putative alpha-ketoglutarate molecule occupies a site physically remote from the proposed binding site for ornithine. Since the structure and charge distribution of the two substrates are largely different, it is conceivable that the substrates of the two half reactions are bound to different residues in separate locations. This hypothesis is consistent with previous reports that the two half reactions displayed different pH optima and that the km of OAT for ornithine and alpha-ketoglutarate followed entirely different pH dependencies.