DESCRIPTION: This proposal involves study of adenosylcobalamin (coenzyme B-12) dependent isomerization of glutamate to 3-methylaspartate catalyzed by glutamate mutase as a model system to investigate two fundamental aspects of enzyme-mediated radical catalysis: i) how do enzymes generate radicals, and ii) how is the removal of hydrogen, the key step in substrate activation, catalyzed? Adenosylcobalamin serves as a source of adenosyl radical that is unmasked by homolysis of its weak cobalt-carbon bond. When bound by the enzyme, a histidine residue coordinates cobalt trans-axially to the cobalt-carbon bond; the histidine, in turn, participates in a hydrogen bond with an aspartate residue. It is hypothesized that the Co-His-Asp triad is important in the binding of the coenzyme and its activation towards homolysis. The effect of mutating the histidine on the enzyme's ability to cleave the cobalt-carbon bond will be investigated. The pK of the histidine will be measured by NMR and the pH dependence of coenzyme binding and catalysis will be examined. Mutants of the aspartate will be made to determine their effect on histidine pK, and to investigate whether changes in pK correlate with changes in enzyme activity and/or coenzyme binding. In the glutamate mutase reaction, the substrate is activated towards rearrangement when a hydrogen atom is removed by the adenosyl radical; after rearrangement, hydrogen is transferred back to give product and regenerate adenosyl radical. Tritiated substrates and coenzyme will be used in stopped-flow experiments to measure the rates of tritium transfer between substrate, coenzyme and product in both directions. Isotope effects will be determined for these steps and hence the rates for hydrogen transfer. By combining these results with steady state kinetic measurements, a free energy profile will be constructed for the complete reaction. An attempt will be made to trap putative reaction intermediates to test mechanistic hypotheses concerning the rearrangement of the substrate radical. X-ray crystallography and protein NMR studies will be initiated to elucidate the three-dimensional structure of the enzyme.