pyrophosphate and orotidylic-acid

The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 +/- 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 +/- 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn2+ and Co2+. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The Km values for the four substrates were determined to be 33 microM for orotate, 64 microM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 microM for orotidine-5-phosphate (OMP), and 36 microM for pyrophosphate. The Km value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.

Topics: Bacterial Proteins; Cations, Divalent; Chromatography, Gel; Cloning, Molecular; Coenzymes; Conserved Sequence; Corynebacterium; Dimerization; Diphosphates; Enzyme Stability; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Lysine; Molecular Sequence Data; Molecular Weight; Orotate Phosphoribosyltransferase; Orotic Acid; Phosphoribosyl Pyrophosphate; Recombinant Proteins; Sequence Analysis, DNA; Uridine Monophosphate

Orotate phosphoribosyltransferase (OPRTase) catalyzes the magnesium-dependent conversion of alpha-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.

Topics: Carbon Radioisotopes; Diphosphates; Hydrogen Bonding; Isotope Labeling; Kinetics; Models, Molecular; Molecular Conformation; Orotate Phosphoribosyltransferase; Protein Structure, Secondary; Salmonella typhimurium; Tritium; Uridine Monophosphate

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)

Topics: Base Sequence; Binding Sites; Computer Graphics; Diphosphates; Enzyme Inhibitors; Enzyme Stability; Kinetics; Lysine; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Orotate Phosphoribosyltransferase; Phosphoribosyl Pyrophosphate; Protein Binding; Protein Structure, Tertiary; Salmonella typhimurium; Substrate Specificity; Uridine Monophosphate

The maximal velocity of the reaction (Vmax) and the half-saturation constant (K0.5) values of the S. typhimurium cytosine deaminase were altered in the presence of its effectors, pyrophosphate and orotidine monophosphate. From the kinetics of orotidine monophosphate inhibition of cytosine deaminase, it was characterized as a mixed-type noncompetitive inhibitor.

Topics: Allosteric Regulation; Bacterial Proteins; Binding, Competitive; Cytosine Deaminase; Diphosphates; Kinetics; Nucleoside Deaminases; Salmonella typhimurium; Uracil Nucleotides; Uridine Monophosphate