The Experts below are selected from a list of 1437 Experts worldwide ranked by ideXlab platform
Charles Grubmeyer - One of the best experts on this subject based on the ideXlab platform.
-
transition state structure of salmonella typhimurium Orotate Phosphoribosyltransferase
Biochemistry, 1996Co-Authors: Charles Grubmeyer, John S BlanchardAbstract:Orotate Phosphoribosyltransferase (OPRTase) catalyzes the magnesium-dependent conversion of α-d-phosphoribosylpyrophosphate (PRPP) and Orotate to orotidine 5‘-monophosphate (OMP) and pyrophosphate. We have determined kinetic isotope effects on the reaction of OMP with pyrophosphate and with the pyrophosphate analog phosphonoacetic acid. In the latter case, full expression of the kinetic isotope effects allowed us to calculate the structure of the transition state for the pyrophosphorylytic reaction. The transition state resembles a classical oxocarbonium ion. Using the recently reported three-dimensional structures of the OPRTase−OMP (Scapin et al., 1994) and the OPRTase−PRPP complexes (Scapin et al., 1995a), we have modeled the calculated transition state structure into the active site of OPRTase. We propose a detailed chemical mechanism which is consistent with these results.
-
The crystal structure of the Orotate Phosphoribosyltransferase complexed with Orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate.
Biochemistry, 1995Co-Authors: Giovanna Scapin, Derya H. Ozturk, Charles Grubmeyer, James C SacchettiniAbstract:The three-dimensional structure of Salmonella typhimurium Orotate Phosphoribosyltransferase (OPRTase) in complex with the ribose 5-phosphate donor alpha-D-5--phosphoribosyl-1-pyrophosphate (PRPP) and the nitrogenous base orotic acid has been solved and refined with X-ray diffraction data extending to 2.3 A resolution to a crystallographic R-factor of 18.7%. The complex was generated by carrying out catalysis in the crystal. Comparison of this structure with the previously reported structure of the orotidine 5'-monophosphate (OMP) complex [Scapin, G., Grubmeyer, C., and Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] revealed that the enzyme backbone undergoes only small movements. The most significant differences occur near the active site, at Ala71-Gly74, with the largest difference involving the side chains of Lys73, Val127-Ala133, the 5'-phosphate binding loop, and a long, solvent-exposed loop at the dimer interface. The position of the ribose moiety is, on the other hand, very different in the OMP and PRPP.Orotate complexes, with its anomeric carbon moving approximately 7 A across the binding cavity. In the PRPP.Orotate complex the highly conserved acidic side chain of Asp124 interacts with the ribose of PRPP, whereas there are no interactions of this aspartate with the substrate in the OMP complex.
-
Locations and functional roles of conserved lysine residues in Salmonella typhimurium Orotate Phosphoribosyltransferase.
Biochemistry, 1995Co-Authors: Derya H. Ozturk, Giovanna Scapin, James C Sacchettini, Ryan H. Dorfman, Charles GrubmeyerAbstract:Salmonella typhimurium Orotate Phosphoribosyltransferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate (OMP) from Orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). There are five highly conserved lysine residues (Lys-19, -26, -73, -100, and -103) in S. typhimurium OPRTase. Here, we report the results of mutagenesis and substrate analog studies to investigate the functional roles of these lysines. Together with information from X-ray crystallography [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754], sequence comparisons, and chemical modification [Grubmeyer, C., Segura, E., & Dorfman, R. (1993) J. Biol. Chem. 268, 20299-20304], this work permits the assignment of functions of the five conserved lysines. Lys-19 is external to the active site, and its mutation to glutamine had little effect on enzyme activity. Lys-26 forms a hydrogen bond to OMP at the 3'-hydroxyl group, and its mutation produced 3-10-fold decreases in kcat. Lys-73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2-hydroxyl and alpha-phosphoryl oxygen of PRPP in their respective substrate complexes. Mutation of Lys-73 produced a 50-100-fold decrease in kcat and an 8-12-fold increase in the KM value for PRPP. Mutation of Lys-100 produced a 5-fold decrease in kcat and a 3-fold increase in the KM for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP.(ABSTRACT TRUNCATED AT 250 WORDS)
-
STRUCTURE AND FUNCTION OF SALMONELLA TYPHIMURIUM Orotate Phosphoribosyltransferase : PROTEIN COMPLEMENTATION REVEALS SHARED ACTIVE SITES
Biochemistry, 1995Co-Authors: Derya H. Ozturk, Giovanna Scapin, James C Sacchettini, Ryan H. Dorfman, Charles GrubmeyerAbstract:A solvent-exposed loop, comprising residues 98-119 of S. typhimurium Orotate Phosphoribosyltransferase (OPRTase), is at the subunit interface of the dimeric enzyme, and its amino acid side chains potentially contact active sites on either subunit. A portion of the loop (103-107) appears to be mobile on the basis of the X-ray structures of enzyme.OMP [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] and enzyme.PRPP.Orotate complexes [Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754]. Lys-103, which is essential for activity [Ozturk, D. H., Dorfman, R. H. Scapin, G., Sacchettini, J. C., & Grubmeyer, C. (1995) Biochemistry 34, 10755-10763], may thus be functional in the active site formed by the adjacent subunit. Asp-125 is an essential residue that is in the middle of the active site. Equimolar mixtures of the nearly inactive K103A and D125N mutant ORPTase subunits produced approximately 21-23% of the enzymatic activity of the wild-type OPRTase. Heterodimer formation in the complemented mixtures was evidenced by various physical methods. Thus, the active site of OPRTase requires Asp-125 from one subunit and Lys-103 from the adjacent subunit. As predicted from the three-dimensional structure, increased activity resulting from complementation was also observed with mixtures of the K103A mutant and the poorly active K73A and K73Q mutants but not with mixtures of D125N and either K73A or K73Q mutants. Neither K103A nor D125N mutants exhibited negative complementation with the wild-type enzyme. A K103A/D125N double mutant enzyme was also constructed and was able to inactivate wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
-
crystal structure of Orotate Phosphoribosyltransferase
Biochemistry, 1994Co-Authors: Giovanna Scapin, Charles Grubmeyer, James C SacchettiniAbstract:Phosphoribosyltransferases (PRTases) are enzymes involved in the synthesis of purine, pyrimidine, and pyridine nucleotides. They utilize α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and a nitrogenous base to form a β-N-riboside monophosphate and pyrophosphate (PP i ), and their functional significance in nucleotide homeostasis is evidenced by the devastating effects of inherited diseases associated with the decreased activity and/or stability of these enzymes. The 2.6-A structure of the Salmonella typhimurium Orotate Phosphoribosyltransferase (OPRTase) complexed with its product orotidine monophosphate (OMP) provides the first detailed image of a member of this group of enzymes
Ronald W. Mcclard - One of the best experts on this subject based on the ideXlab platform.
-
Ternary complex formation and induced asymmetry in Orotate Phosphoribosyltransferase.
Biochemistry, 2007Co-Authors: Lilian González-segura, John F. Witte, Ronald W. Mcclard, Thomas D HurleyAbstract:Orotate Phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-alpha-d-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5'-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 A resolution); a ternary complex of enzyme, Mg2+-PRPP, and OA (1.74 A resolution); and the binary product complex of enzyme with OMP (1.89 A resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5 degrees in the OMP structure and an average of 24.6 degrees in the OA/Mg2+-PRPP ternary complex. As expected, the conserved loop, composed of residues 104-116, moves extensively and adopts a single stable conformation during the catalytic cycle in order to sequester the substrates from bulk solvent in the ternary complex. The OA and Mg2+-PRPP molecules bound in the ternary complex are oriented for proper attack of the N1 atom of OA onto the C1 atom of the ribose ring. This orientation of substrates, combined with the positioning of the flexible loop, provides a clear picture of a catalytically poised reaction complex for type I Phosphoribosyltransferases. The structural asymmetry present in these structures, as well as that found in a recent structure of the S. typhimurium enzyme, combined with the closure of the flexible loop from one subunit into the active site of the opposing subunit in the ternary complex is consistent with the kinetic data [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342] that demonstrate induced nonequivalence and cooperativity of OPRTase.
-
ternary complex formation and induced asymmetry in Orotate Phosphoribosyltransferase
Biochemistry, 2007Co-Authors: John F. Witte, Ronald W. Mcclard, Lilian Gonzalezsegura, Thomas D HurleyAbstract:Orotate Phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-α-d-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5‘-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 A resolution); a ternary complex of enzyme, Mg2+-PRPP, and OA (1.74 A resolution); and the binary product complex of enzyme with OMP (1.89 A resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5° in the OMP struct...
-
'Irreversible' slow-onset inhibition of Orotate Phosphoribosyltransferase by an amidrazone phosphate transition-state mimic.
Bioorganic & medicinal chemistry letters, 2006Co-Authors: John F. Witte, Kathryn E. Bray, Chelsea K. Thornburg, Ronald W. McclardAbstract:A mimic of the putative transition-state intermediate has been synthesized and found to be a very slow-onset inhibitor of yeast Orotate Phosphoribosyltransferase. The mechanism of inhibition may involve a rate-determining isomerization of the enzyme to a form receptive to the inhibitor, which then remains tightly bound.
-
Cloning, overproduction, and purification of native and mutant recombinant yeast Orotate Phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetim
Archives of biochemistry and biophysics, 1999Co-Authors: John F. Witte, Raymond Tsou, Ronald W. McclardAbstract:Abstract The gene for Orotate Phosphoribosyltransferase from Saccharomyces cerevisiae has been subcloned into an Escherichia coli overexpression vector and the enzyme has been produced in large quantities, thus simplifying the purification to one step. We were able to repeat the published (J. Victor, L. B. Greenberg, and D. L. Sloan J. Biol. Chem. 254, 2647–2655, 1979). 32 PP i /5-phosphorylribose 1-α-diphosphate exchange experiments and could demonstrate the exchange by magnetization inversion transfer NMR experiments as well. However, when contaminating orotidine 5′-monophosphate (OMP) was eliminated with OMP decarboxylase, any evidence of magnetization transfer vanished. Consequently, it is concluded that a ping pong mechanism is not operable and that a previously proposed oxocarbocation intermediate along the pathway to OMP does not persist long enough in the catalytic cycle of this enzyme to be recognized by NMR exchange experiments.
Nobuo Tanaka - One of the best experts on this subject based on the ideXlab platform.
-
Medium temperature, 310 K, provides single crystals of Orotate Phosphoribosyltransferase from Thermus thermophilus.
Acta Crystallographica Section D Biological Crystallography, 1999Co-Authors: Hiroshi Hamana, Takao Shinozawa, Hideaki Moriyama, Nobuo TanakaAbstract:Crystallization of Orotate Phosphoribosyltransferase from a thermophilic organism, Thermus thermophilus, was achieved using the hanging-drop vapour-diffusion method coupled with a macroseeding starter. Small needle-like microcrystals were grown in a fresh protein solution in the presence of 2-methyl-2,4-pentanediol at 298 K or below. Although these normal temperature conditions caused stacking crystallization, an increase of temperature to 310 K permitted crystal growth. This was because of increased enzyme solubility at the higher temperature. The crystal was found to belong to the monoclinic space group P21 with unit-cell parameters a = 44.4, b = 59.6, c = 67.8 A and β = 98.3°.
-
Medium temperature, 310 K, provides single crystals of Orotate Phosphoribosyltransferase from Thermus thermophilus.
Acta crystallographica. Section D Biological crystallography, 1999Co-Authors: Hiroshi Hamana, Takao Shinozawa, Hideaki Moriyama, Nobuo TanakaAbstract:Crystallization of Orotate Phosphoribosyltransferase from a thermophilic organism, Thermus thermophilus, was achieved using the hanging-drop vapour-diffusion method coupled with a macroseeding starter. Small needle-like microcrystals were grown in a fresh protein solution in the presence of 2-methyl-2,4-pentanediol at 298 K or below. Although these normal temperature conditions caused stacking crystallization, an increase of temperature to 310 K permitted crystal growth. This was because of increased enzyme solubility at the higher temperature. The crystal was found to belong to the monoclinic space group P21with unit-cell parameters a = 44.4, b = 59.6, c = 67.8 A and beta = 98.3 degrees.
Masakazu Fukushima - One of the best experts on this subject based on the ideXlab platform.
-
Orotate Phosphoribosyltransferase expression level in tumors is a potential determinant of the efficacy of 5 fluorouracil
Biochemical and Biophysical Research Communications, 2007Co-Authors: Etsuko Sakamoto, Hideki Nagase, Takashi Kobunai, Masakazu FukushimaAbstract:Although the intratumoral expression levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are known to affect the antitumor activity of 5-fluorouracil (5-FU), the importance of Orotate Phosphoribosyltransferase (OPRT) has remained unclear. This study investigated the relationship between intratumoral OPRT expression and the antitumor activity of 5-FU using human NCI60 cell lines with similar levels of TS and DPD messenger RNAs, as well as 31 tumor xenografts. The OPRT mRNA level was positively correlated with the 5-FU efficacy in these cell lines. In vitro, the 50% growth-inhibitory concentrations of 5-FU were closely correlated with the OPRT mRNA levels in cancer cell lines with similar levels of TS mRNAs when combined with a DPD inhibitor. Moreover, downregulation of OPRT with small-interfering RNA decreased the sensitivities of the cultured tumor cells to 5-FU. These results suggest that the OPRT expression level in tumors is an additional determinant of the efficacy of 5-FU.
-
the expression profiles of Orotate Phosphoribosyltransferase and dihydropyrimidine dehydrogenase in gastric cancer and their clinical significance
Oncology Reports, 2006Co-Authors: Mamoru Oeda, Yuichi Sanada, Kazuhiro Yoshida, Takahisa Suzuki, Yoshiyuki Wada, Hirozumi Mizuiri, Kazuo Konishi, Hideo Shigematsu, Kazuaki Tanabe, Masakazu FukushimaAbstract:Orotate Phosphoribosyltransferase (OPRT) is an enzyme that causes the activation of 5-fluorouracil (5-FU). Dihydropyrimidine dehydrogenase (DPD) is known to catabolize 5-FU, which is widely used in chemotherapeutic treatments for patients with a variety of malignant tumors including gastric and colorectal cancer. The expression and activities of these two enzymes therefore play important roles in the response of cancer patients to chemotherapy. However, little is known about the expression of these enzymes in gastric cancer. In the present study, we further elucidate the expression patterns of ORPT and DPD and their clinicopathological significance by immunohistochemical analysis in 221 and RT-PCR in 36 gastric cancer samples. The expression of OPRT by immunohistochemical analysis was detected in 117 (52.9%) cases, whereas DPD was detected in 66 (29.9%) cases. Moreover, the level of expression of OPRT was found to correlate with the depth of tumor invasion and a poorer prognosis. Although the mRNA and protein expression of OPRT and DPD levels did not correlate, an inverse correlation in the expression of OPRT and DPD was observed by RT-PCR. The survival benefit of post-operative adjuvant chemotherapy could not be confirmed in our present analysis. However, among the patients who had received such treatment with 5-FU or its derivatives, the prognosis in cases with low DPD levels was better than that in cases with high DPD expression by immunohistochemical analysis. These results indicate that the expression of OPRT and DPD are important predictors of both survival and the response to adjuvant chemotherapy in gastric cancer patients.
-
population study of thymidylate synthase dihydropyrimidine dehydrogenase and Orotate Phosphoribosyltransferase in patients with solid tumors
Cancer Research, 2006Co-Authors: Masakazu Fukushima, Yousuke Fukui, Yoshikazu Sugimoto, Sekio NagayamaAbstract:3639 Purpose: Thymidylate synthase(TS), dihydropyrimidine dehydrogenase(DPD) and Orotate Phosphoribosyltransferase(OPRT) has been suggested to be predictive factors of 5-fluorouracil(5FU) therapy in cancer patients. To determine the differences in expression levels of these enzymes in solid tumors, we carried out a large-scale population study of the mRNA expression, enzyme content and enzyme activity of TS, DPD and OPRT. Methods: Surgical specimens were obtained from collaborating universities and hospitals in Japan. A total of 29957 clinical dissected specimens, consisting 4871 for mRNA, 13433 for enzyme amount, 11653 for enzyme activity, were collected between July 1998 and June 2005. The expression of TS, DPD and OPRT mRNA were assayed by Danenberg tumor profile (DTP). Enzyme amount of TS, DPD and OPRT were measured by enzyme linked immunosorbent assay (ELISA). TS activity and DPD activity of collected specimens were analyzed by [ 3 H]-FdUMP binding assay and radio-enzymatic assay used [ 14 C]-5-FU, respectively. Results: TS expression in the gastric cancer was significantly higher than matched non-cancerous tissue in mRNA, amount and activity. DPD expressions in the gastric, breast and lung cancer were significantly higher than matched non-cancerous tissues in amount and activity. We classified each type of carcinoma into four categories by using the median TS and DPD levels of all tumors as cut off value. About 60% of the population of gastric cancer had high DPD in the expression of mRNA, enzyme amount and activity, and about 65% of colorectal cancer had low DPD population. About 50% to 70% of the lung and pancreas cancer patients were distributed in low TS/high DPD group. There was no correlation between TS and DPD expression, so TS and DPD in tumors seemed to be independent factor. However, the TS expression of mRNA was correlated with OPRT expression (Spearman’s correlation coefficient = 0.42, p
-
preparation of anti Orotate Phosphoribosyltransferase antibody and its application to immunochemical detection in human tumor cells
International Journal of Molecular Medicine, 2005Co-Authors: Kazuki Sakamoto, Yoshikazu Sugimoto, Kazutaka Miyadera, Masakazu FukushimaAbstract:Orotate Phosphoribosyltransferase (OPRT) is a key enzyme in the anabolism of 5-fluorouracil (5-FU), and its expression in tumors is thought to increase the efficacy of 5-FU against the tumor. To detect the OPRT protein byimmunoblot and/or immunohistochemical methods, we prepared highly specific antibody to the peptides contained in the human OPRT amino acid sequence. The anti-OPRT polyclonal antibodies, obtained by immunizing rabbits with the OPRT peptides, had a high specificity for the OPRT protein in human tumor xenografts when it was analyzed by immunoblotting, and furthermore, there was a positive correlation between OPRT activity and protein content in 12 human tumors (R 2 =0.632). These results suggest that immunohistochemical detection of tumoral OPRT protein expression with our anti-OPRT antibodies may provide a useful method for predicting the clinical response to 5-FU-based chemotherapy.
-
heat assisted stretching of paraffin sections on hot plate weakens immunoreactivity of Orotate Phosphoribosyltransferase
Acta Histochemica Et Cytochemica, 2005Co-Authors: Shingo Kamoshida, Masakazu Fukushima, Yoshikazu Sugimoto, Yoichi Sakurai, Kazuki Sakamoto, Nao Sakamoto, Hiroshi Matsuoka, Yutaka TsutsumiAbstract:Orotate Phosphoribosyltransferase (OPRT) is the key enzyme for the phosphorylation of 5-fluorouracil (5-FU), the rate-limiting step for acquiring its anti-tumor effect. Since high enzyme activities and mRNA levels of OPRT are said to be associated with 5-FU chemosensitivity of cancer cells, the immunohistochemical demonstration of OPRT can be expected to contribute to the selection of patients who suffer from 5-FU-sensitive cancer. During a study for establishing the appropriate immunostaining condition using rabbit antiserum in formalin-fixed, paraffin-embedded sections of human cancer, we unexpectedly uncovered the fact that heat-assisted stretching of paraffin sections on a hot plate just after sectioning was critical for preserving OPRT immunoreactivity; namely, stretching sections briefly at 70°C or higher significantly reduced the immunoreactivity. Overnight drying of the sections in an oven at 37°C or 60°C did not influence the immunoreactivity, but pretreatments, including 0.2% trypsin, 0.002% proteinase K, and pressure cooking in 10 mM citrate buffer, pH 6.0 and pH 7.0, and 1 mM ethylenediaminetetraacetic acid solution, pH 8.0, seriously deteriorated the OPRT epitopes. The immunoreactivity was relatively resistant to overfixation, though weakened after fixation in formalin for four weeks. Xenografts with high OPRT enzyme activities showed distinct positive cytoplasmic staining, but those with low OPRT activities were equivocal. OPRT expression in routinely processed cancer tissues also provided valuable data.
John F. Witte - One of the best experts on this subject based on the ideXlab platform.
-
Ternary complex formation and induced asymmetry in Orotate Phosphoribosyltransferase.
Biochemistry, 2007Co-Authors: Lilian González-segura, John F. Witte, Ronald W. Mcclard, Thomas D HurleyAbstract:Orotate Phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-alpha-d-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5'-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 A resolution); a ternary complex of enzyme, Mg2+-PRPP, and OA (1.74 A resolution); and the binary product complex of enzyme with OMP (1.89 A resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5 degrees in the OMP structure and an average of 24.6 degrees in the OA/Mg2+-PRPP ternary complex. As expected, the conserved loop, composed of residues 104-116, moves extensively and adopts a single stable conformation during the catalytic cycle in order to sequester the substrates from bulk solvent in the ternary complex. The OA and Mg2+-PRPP molecules bound in the ternary complex are oriented for proper attack of the N1 atom of OA onto the C1 atom of the ribose ring. This orientation of substrates, combined with the positioning of the flexible loop, provides a clear picture of a catalytically poised reaction complex for type I Phosphoribosyltransferases. The structural asymmetry present in these structures, as well as that found in a recent structure of the S. typhimurium enzyme, combined with the closure of the flexible loop from one subunit into the active site of the opposing subunit in the ternary complex is consistent with the kinetic data [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342] that demonstrate induced nonequivalence and cooperativity of OPRTase.
-
ternary complex formation and induced asymmetry in Orotate Phosphoribosyltransferase
Biochemistry, 2007Co-Authors: John F. Witte, Ronald W. Mcclard, Lilian Gonzalezsegura, Thomas D HurleyAbstract:Orotate Phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-α-d-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5‘-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 A resolution); a ternary complex of enzyme, Mg2+-PRPP, and OA (1.74 A resolution); and the binary product complex of enzyme with OMP (1.89 A resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5° in the OMP struct...
-
'Irreversible' slow-onset inhibition of Orotate Phosphoribosyltransferase by an amidrazone phosphate transition-state mimic.
Bioorganic & medicinal chemistry letters, 2006Co-Authors: John F. Witte, Kathryn E. Bray, Chelsea K. Thornburg, Ronald W. McclardAbstract:A mimic of the putative transition-state intermediate has been synthesized and found to be a very slow-onset inhibitor of yeast Orotate Phosphoribosyltransferase. The mechanism of inhibition may involve a rate-determining isomerization of the enzyme to a form receptive to the inhibitor, which then remains tightly bound.
-
Cloning, overproduction, and purification of native and mutant recombinant yeast Orotate Phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetim
Archives of biochemistry and biophysics, 1999Co-Authors: John F. Witte, Raymond Tsou, Ronald W. McclardAbstract:Abstract The gene for Orotate Phosphoribosyltransferase from Saccharomyces cerevisiae has been subcloned into an Escherichia coli overexpression vector and the enzyme has been produced in large quantities, thus simplifying the purification to one step. We were able to repeat the published (J. Victor, L. B. Greenberg, and D. L. Sloan J. Biol. Chem. 254, 2647–2655, 1979). 32 PP i /5-phosphorylribose 1-α-diphosphate exchange experiments and could demonstrate the exchange by magnetization inversion transfer NMR experiments as well. However, when contaminating orotidine 5′-monophosphate (OMP) was eliminated with OMP decarboxylase, any evidence of magnetization transfer vanished. Consequently, it is concluded that a ping pong mechanism is not operable and that a previously proposed oxocarbocation intermediate along the pathway to OMP does not persist long enough in the catalytic cycle of this enzyme to be recognized by NMR exchange experiments.