Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium.
The chemical mechanism of the phosphoribosyltransferases (PRTases), although largely unknown, may proceed either via a concerted direct-transfer mechanism or with a two-step mechanism involving a carboxonium-like intermediate. To study this question, we have cloned the Salmonella typhimurium pyrE gene, coding for the enzyme orotate phosphoribosyltransferase (EC 2.2.4.10, OPRTase), and developed a bacterial strain that overproduces the enzyme, which we have purified to homogeneity. Initial velocity and product inhibition studies indicated that S. typhimurium OPRTase follows a random sequential kinetic mechanism. This result was further confirmed by equilibrium isotope exchange studies on two substrate-product pairs, PRPP-PPi and OMP-orotate. In addition, the rates of the individual equilibrium isotope exchanges allowed us to conclude that PPi release and PRPP release were the rate-determining steps in the forward and reverse reactions, respectively. Although partial reactions between the two substrate-product pairs, PRPP-PPi and OMP-orotate, were observed, further studies revealed that these exchanges were a result of contaminations. Our results are significant in that S. typhimurium OPRTase, like most PRTases but in contrast to its yeast homologue, follows sequential kinetics. The artifactual partial isotope exchanges found in this work may have implications for similar prior work on the yeast enzyme. In view of the careful isotope effect studies of Parsons and co-workers [Goitein, R.K., Chelsky, D., & Parsons, S.M. (1978) J. Biol. Chem. 253, 2963-2971] and the results obtained by us, we propose that PRTases may involve a direct-transfer mechanism but with low bond order to the leaving pyrophosphate moiety and attacking base.
Bhatia
M B
MB
Department of Biology, New York University, New York 10003.
Vinitsky
A
A
Grubmeyer
C
C
D002250:Carbon Radioisotopes; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D007700:Kinetics; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009963:Orotic Acid / Q000378:metabolism; D010957:Plasmids; D011865:Radioisotope Dilution Technique; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000201:enzymology*
D002250:Carbon Radioisotopes; D009963:Orotic Acid; D009962:Orotate Phosphoribosyltransferase