Unidirectional Steady State Rates of Central Metabolism Enzymes Measured Simultaneously in a Living Plant Tissue

Abstract

The unidirectional steady state reaction rates of several enzymes and metabolic fluxes of distinct processes were measured simultaneously in hypoxic maize root tips using two-dimensional phosphorus NMR exchange spectroscopy. A single spectrum monitors ATP synthesis and hydrolysis as well as the activities of four enzymes involved in key pathways of central metabolism: UDP-glucose pyrophosphorylase, phosphoglucomutase, hexose-phosphate isomerase, and enolase. The corresponding unidirectional reaction rates and net metabolic fluxes were calculated from spectral intensities. This method provides a unique picture, at enzyme resolution, of how metabolism reacts in a concerted fashion to changes in external parameters such as temperature and oxygen concentration. By increasing hypoxia via an increase in temperature, we measured the expected increase in glycolysis through enolase activity while total ATP synthesis settled. At the same time, we observed a net flux through phosphoglucomutase and UDP-glucose pyrophosphorylase toward carbohydrate synthesis. This result is discussed in relation to the current hypothesis on the turnover of cell walls and sucrose. This reaction also produces a net flux of pyrophosphate, which is needed by pyrophosphate:fructose-6-phosphate 1-phosphotransferase to work as a glycolytic enzyme.