In cancer and autoimmune diseases, rapid cell division depends on nucleotide biosynthesis rather than the salvage pathways that predominate in normal cells. Thus, mechanism-based inhibitors of orotidine 5-phosphate decarboxylase (ODCase), the last step in the formation of pyrimidine nucleotides, may have therapeutic value in the treatment of these diseases, and as antibacterial and antimalarial agents. We will use kinetic and mass spectrometric measurements to test the possibility that the imperfect binding of the transition state analogue 6-hydroxy-UMP arises from the trapping of a water molecule in its inhibitory complex with ODCase, in a gap deep in the active site that is believed to be occupied by CO2 released during decarboxylation. Based on recent observations of the binding affinities of 6-C(=S)NH2 and 6-C(=Se)NH2 derivatives of UMP, we will also prepare new 6-substituted inhibitors that are expected to fill that gap and bear a closer resemblance to the altered substrate in the transition state, in which the C-C bond is stretched but not broken. Preliminary experiments in this laboratory indicate that uroporphyrin III decarboxylase (UroD), which is missing in most patients with porphyria of the most common type, generates one of the largest rate enhancement that has been observed for any enzyme. Unlike ODCase, UroD is almost devoid of active site residues that might be involved in catalysis. We propose to test the possibility that 2 essential arginine residues play a direct role in catalysis, by stabilizing the carbanion generated by the departure of CO2, then furnishing the proton that takes its place;and that this reaction represents an extreme example of catalysis by desolvation. These analogues are expected to be useful, in conjunction with exact structural methods, in working out the detailed mechanism of catalysis by this enzyme. Model experiments will also be conducted to determine the susceptibility of decarboxylation to catalysis by desolvation, and to catalysis by molecules representing groups that are present at the human enzyme's active site.
In cancer and autoimmune diseases, rapid cell division depends on nucleotide biosynthesis rather than the salvage pathways that predominate in normal cells. This project involves the design of mechanism-based inhibitors of orotidine 5- phosphate decarboxylase (ODCase), the last step in the formation of pyrimidine nucleotides, may have therapeutic value in the treatment of these diseases, and as antibacterial and antimalarial agents. This project also aims to establish the mechanism of action of uroporphyrin III decarboxylase, whose deficiency is responsible for the most common form of porphyria. There is reason to believe that this reaction, which occurs extremely slowly in the absence of enzyme (half- life 25,000 years) may represent an extreme example of catalysis by desolvation;if that is found to be the case, then there are reasonable prospects of developing artificial catalysts to alleviate the condition.
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