AN APPROACH TO THE IN-VITRO STUDY OF THE UTP UDPGLUCOSE/UDP MOIETY-CONSERVED CYCLE/

The kinetic behavior of a moiety-conserved ternary cycle is tested exp erimentally. This system contains the enzymes UDPglucose pyrophosphory lase, glycogen synthase and nucleoside diphosphokinase, converting res pectively UTP into UDPglucose, then into UDP and back to UTP in a cycl ic manner. The UDPGlc P(2)ase and NDPK steps are made irreversible by addition of inorganic pyrophosphatase and phosphocreatine kinase, resp ectively. In order to predict both the evolution and the steady-state values of the various substrates, a model is derived, which takes into account the actual enzyme rate expressions and parameter values, as d etermined under our experimental conditions. In that model, the UTP, U DPglucose and UDP are taken as the variables, whereas the total concen tration of the substrate pool and the four enzyme maximal activities a re chosen as the control parameters. Depending upon the various parame ter values, monostability, reversible bistability and irreversible tra nsitions may theoretically occur. However, it turns out that some of t hese values for which multistability might occur, are not accessible e xperimentally. Under conditions of monostability, the evolutions of th e three substrates as experimentally measured are shown to be in good qualitative and quantitative agreement with the model predictions. The relaxation times between two consecutive steady states when a paramet er is varied, are shown to be long-lasting processes (several hours). That such an experimental ternary substrate cycle actually exhibits a low sensitivity to any perturbation, addresses the issue to knowing if the same property is likely to occur in vivo, or, in other words, do large moiety-conserved cycles act as metabolic buffers?