In VivoDynamics of the Pentose Phosphate Pathway inSaccharomyces cerevisiae

Abstract

Thein vivodynamics of the pentose phosphate pathway has been studied with transient experiments in continuous culture ofSaccaromyces cerevisiae. Rapid sampling was performed with a special sampling device after disturbing the steady state with a pulse of glucose. The time span of observation was 120 s after the pulse. During this short time period the dynamic effect of protein biosynthesis can be neglected. The metabolites of interest (glu- cose 6-phosphate, NADP, NADPH, 6-phosphogluconate, and MgATP2−) we determined with enzymatic assays and HPLC. The experimental observations were then used for the identification of kinetic rate equations and parameters underin vivoconditions. In accordance with results fromin vitrostudies thein vivodiagnosis supports an ordered Bi-Bi mechanism with noncompetitive inhibition by MgATP2−for the enzyme glucose-6-phosphate dehydrogenase. In the case of 6-phosphogluconate dehydrogenase an ordered Bi-Ter mechanism with a competitive inhibition by MgATP2−has been found. Because the MgATP2−concentration decreases abruptly after the pulse of glucose the inhibitory effect vanishes and the flux through the pentose phosphate pathway increases. This regulation phenomenon guarantees the balance of fluxes through glycolysis and pentose phosphate pathway during the dynamic time period.