The overall goal is to provide a molecular understanding of enzymic mechanism of Prostaglandin H synthase (PGHS). The working model is a free radical branched chain mechanism in which a tyrosine radical, generated after interaction of the synthase heme with hydroperoxide in the peroxidase catalytic cycle, serves a central catalytic role in the propagation of the cyclooxygenase reaction. To test this mechanism, we propose to: 1. Investigate the structure-function relationship between the heme prosthetic group and the apoprotein. Heme-binding mechanism will be studied using several spectroscopic methods accompanied with enzyme activity analysis. A series of modified porphyrins and porphyrins coordinated with metal ions other than iron will be used as probes to react with the apoprotein for this study. Heme binding stoichiometry will be assesed by first a horizontal comparison of the heme titration (or binding) using apo PGHS samples prepared from different procedures, followed by a vertical comparison of methodological differences used to measure the heme stoichiometry. 2. Characterize the detailed mechanism of each enzyme activity, i.e., cyclooxygenase and peroxidase. We propose to use transient kinetic measurements to obtain correlated data of important reaction intermediates which can be monitored by optical, EPR or isotope tracer. Arachidonate, substrate for the cyclooxygensase, and a series of peroxides including hydrogen peroxide, ethyl hydroperoxide, PCTG2 and various positional isomers of hydroperoxy-eicosateraenoic acid will be examined systematically. 3. Identify the functionally critical amino acid residues in this hemeprotein. Using a human cDNA clone and site-directed mutagenesis, we will specifically test the influence of mutation of a specific tyrosine, a pair of histidine and a serine residue on the enzyme activity. The tyrosine residue was proposed to be the linker between the two enzyme activities which is first generated by the peroxidase activity and then acts as an activator for the cyclooxygenase, the' histidine pair is proposed to be the axial ligands for the heme iron through EPR studies; and the serine is identified as the acetylation site of aspirin. The first specific approach helps to solve the controversy of heme stoichiometry and to gain knowledge about the details of the interaction between heme and the apoprotein. the second study by transient kinetics will reveal the detail reaction sequence of PGHS and will help resolve the current conflict of the observed temporal events of enzyme catalysis. The third and final approach of site-directed mutagenesis will help to pinpoint the important amino acid residues and Will enhance the elucidation of the overall enzymic reaction mechanism.
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