Quinolinate phosphoribosyltransferase: kinetic mechanism for a type II PRTase.
Quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) catalyzes the formation of nicotinate mononucleotide, carbon dioxide, and pyrophosphate from 5-phosphoribosyl 1-pyrophosphate (PRPP) and quinolinic acid (QA, pyridine 2,3-dicarboxylic acid). The enzyme is the only type II PRTase whose X-ray structure is known. Here we determined the kinetic mechanism of the enzyme from Salmonella typhimurium. Equilibrium binding studies show that PRPP and QA each form binary complexes with the enzyme, with K(D) values (53 and 21 microM, respectively) similar to their K(M) values (30 and 25 microM, respectively). Although neither PP(i) nor NAMN products bound well to the enzyme, 130-fold tighter binding of PP(i) (K(D) = 75 microM) and NAMN (K(D) = 6 microM) in a ternary complex was observed. Phthalic acid (K(D) = 21 microM) and PRPP each caused a 2.5-fold tightening of the other's binding. Isotope trapping experiments indicated that the E.QA complex is catalytically competent, whereas the E.PRPP complex could not be trapped. Pre-steady-state kinetics gave a linear rate of NAMN formation, indicating that on-enzyme phosphoribosyl transfer chemistry is rate-determining. Isotope trapping from the steady state revealed that nearly all QA and about one-third of PRPP in ternary enzyme.QA.PRPP complexes could be trapped as the product. Substrate inhibition by PRPP was observed. These data demonstrate a predominantly ordered kinetic mechanism in which productive binding of quinolinic acid precedes that of PRPP. An E.PRPP complex exists as a nonproductive side branch.
Cao
Hong
H
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, PA 19140, USA.
Pietrak
Beth L
BL
Grubmeyer
Charles
C
D007554:Isotopes; D007700:Kinetics; D010430:Pentosyltransferases / Q000378:metabolism*; D010795:Phthalic Acids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)
D007554:Isotopes; D010795:Phthalic Acids; D011994:Recombinant Proteins; C032279:phthalic acid; D010430:Pentosyltransferases; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)