A continuation of research on the catalytic mechanism of the phosphoglucomutase reaction is proposed that includes X-ray diffraction studies on the crystalline enzyme, as well as NMR (1H, 13C, 31P, and 113Cd), kinetic, and isotopic rate studies on solutions of the enzyme. These studies will focus on the mechanism that the enzyme uses to produce its large, catalytic rate effect. An electron density map with a nominal resolution of 2.7 Angstrom unit will be improved by molecular replacement techniques; the current molecular model will be modified and refined. Phases from the model plus additional diffraction data will be used to construct difference density maps of enzyme- substrate analog complexes after completing an evaluation of several possible complexes of this types, partly via NMR techniques. Related studies will explore 1H/1H interactions between bound substrate and the enzyme in substrate analog complexes. An attempt also will be made to observe and evaluate 1H/113Cd interactions between the bound metal ion and bound substrate. 31P-NMR studies on the interactions between the Cd2+ complex of the dephospho-enzyme and substrate analogs such as straight-chain 1,4-diol bisphosphates will be continued to further define how G1c-1, 6-P2 is bound by this form of the enzyme so that either the 1- or the 6-phosphate group is in position for transfer to a single acceptor group on the enzyme. A series of kinetic studies will be conducted with butane-1,4-diol monophosphate, which acts as an inefficient substrate (phosphate acceptor) to isolate the effects of pH and identity of the activating metal ion on the rate-controlling step in this process: the bond making/breaking step. A determination of the (18 O) kinetic isotope effect on the reaction (in the reverse direction, where (18 O)-labelled butane-1,4-diol-P2 is used to phosphorylate the enzyme) also will be conducted. Studies on how cosolutes affect the crystal growth of phosphoglucomutase at high salt will be continued by comparing the effects of polyethylene glycol and detergents on crystal growth rate. Procedures for reducing the salt content of such crystals to aid in the binding of substrates and analogs will be explored. Studies of how conditions used for data collection affect the quality of X-ray diffraction data also will be conducted.
| Chibucos, Marcus C; Zweifel, Adrienne E; Herrera, Jonathan C et al. (2014) An ontology for microbial phenotypes. BMC Microbiol 14:294 |
