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 Lifetime

A Nonradioactive High-Performance Liquid Chromatographic Microassay for Uridine 5′-Monophosphate Synthase, Orotate Phosphoribosyltransferase, and Orotidine 5′-Monophosphate Decarboxylase

“…Although these assays are suitable when there are large numbers and volumes of the samples, they have drawbacks when high sensitivity is needed. In addition, the HPLC methods are at least 100-fold more sensitive than spectrophotometric assays (12)(13)(14)(15)(16)(17). Our HPLC methods are also comparable with other HPLC methods using different conditions for both enzymatic and HPLC assay for measurement of low UMPS activity of human red cells (10,11).…”

“…Two highperformance liquid chromatographic (HPLC) assays using nonradiochemical method have been used for measurement of the very low UMPS activities of human chorionic villus and red cell (10,11). Most of the monofunctional OPRTase and ODCase assays are based on spectrophotometric quantitation of orotate conversion to OMP and OMP conversion to UMP at 295 and 285 nm, respectively (12)(13)(14)(15)(16)(17).…”

Kinetic Studies of the Uracil Phosphoribosyltransferase Reaction Catalyzed by the Bacillus subtilis Pyrimidine Attenuation Regulatory Protein PyrR

“…A classical Ping Pong mechanism predicts the existence of a free phosphoribosyl-enzyme intermediate and capacity to catalyze exchange reactions between reactant pairs in the absence of co-substrates. These properties have been reported for various PRTases (27,33), but on further scrutiny the conclusions were invalidated (28,34,35). In no case has persuasive evidence for a classic Ping Pong mechanism been put forward for a Type I PRTase.…”

An organism-independent unified model for activity of orotate phosphoribosyltransferases for orotidine monophosphate synthesis

“…The non-existence of ping-pong bi-bi mechanism proposed by Bhatia et al was further supported by a study by Witte et al (1999). The authors observed that it was not possible to carry out the reaction as two independent half reactions, a requirement for ping-pong bi-bi mechanism.…”

“…There are several issues concerning the mechanism of the reaction which remain unresolved till date and require attention. Experimental studies have been carried out probing the mechanism of OPRT-action (Victor et al, 1979a,b;Syed et al, 1987;Bhatia et al, 1990;Tao et al, 1996;Witte et al, 1999;McClard et al, 2006;Krungkrai et al, 2004). Surprisingly, different studies either report different mechanisms for the reaction which are not in agreement or the mechanisms are suspected to be organism-dependent thereby making the mechanism of the reaction doubtful.…”