Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).
The presence of a proton-donating catalytic amino acid side chain in orotidylate decarboxylase (ODCase) was sought by site-directed mutagenesis. Replacement of yeast ODCase Lys93 with a cysteine resulted in a mutant protein (K93C) with no measurable activity, representing a decrease in activity by a factor of, at most, 2 x 10(-8) times the activity of the wild-type enzyme. Treatment of this mutant protein with 2-bromoethylamine, designed to append Cys93 to yield S-(2-aminoethyl)cysteine, restored activity by a factor of at least 5 x 10(5) over the untreated mutant protein. Activity could not be restored by treatment with other brominated reagents designed to replace the epsilon-amino of S-(2-aminoethyl)Cys93 with a different functional group. The overall architecture of the K93C protein was not significantly changed, as judged by the similar dimerization properties (in the absence of ligands) of the mutant enzyme compared to the wild-type enzyme. The binding affinity of the substrate orotidylate was not measurably changed by the mutation, indicating that Lys93 has an essential role in catalysis which is mechanistically distinguishable from substrate binding. Apparently the mutation removes an integral portion of the active site and does not drastically affect the structural or substrate binding properties. However, the affinities of the mutant protein for the competitive inhibitors 6-azauridylate (6-azaUMP) and UMP are significantly altered from the pattern seen with the wild-type enzyme. The K93C protein has an affinity for the neutral ligand UMP which is greater than that for the anionic 6-azaUMP, in clear contrast to the preference for 6-azaUMP displayed by the wild-type enzyme. Lys93 is apparently critical for catalysis of the substrate to product and for the binding of anionic inhibitors; the data are discussed in terms of previously existing models for transition-state analogue inhibitor binding and catalysis.
Human malaria parasite, Plasmodium falciparum, can only synthesize pyrimidine nucleotides using the de novo pathway, whereas mammalian cells obtain pyrimidine nucleotides from both the de novo and salvage pathways. The parasite's orotate phosphoribosyltransferase (PfOPRT) and orotidine 5'-monophosphate decarboxylase (PfOMPDC) of the de novo pyrimidine pathway are attractive targets for antimalarial drug development. Previously, we have reported that the two enzymes in P. falciparum exist as a multienzyme complex containing two subunits each of 33-kDa PfOPRT and 38-kDa PfOMPDC. In this report, the gene encoding PfOPRT has been cloned and expressed in Escherichia coli. An open reading frame of PfOMPDC gene was identified in the malaria genome database, and PfOMPDC was cloned from P. falciparum cDNA, functionally expressed in E. coli, purified, and characterized. The protein sequence has <20% identity with human OMPDC and four microbial OMPDC for which crystal structures are known. Recombinant PfOMPDC was catalytically active in a dimeric form. Both recombinant PfOPRT and PfOMPDC monofunctional enzymes were kinetically different from the native multienzyme complex purified from P. falciparum. Oligomerization of PfOPRT and PfOMPDC cross-linked by dimethyl suberimidate indicated that they were tightly associated as the heterotetrameric 140-kDa complex, (PfOPRT)2(PfOMPDC)2. Kinetic analysis of the PfOPRT-PfOMPDC associated complex was similar to that of the native P. falciparum enzymes and was different from that of the bifunctional human enzymes. Interestingly, a nanomolar inhibitor of the yeast OMPDC, 6-thiocarboxamido-uridine 5'-monophosphate, was about 5 orders of magnitude less effective on the PfOMPDC than on the yeast enzyme. Our results support that the malaria parasite has unique structural and functional properties, sharing characteristics of the monofunctional pyrimidine-metabolizing enzymes in prokaryotes and bifunctional complexes in eukaryotes.
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