Enzymology in vivo using NMR and molecular genetics

Abstract

Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured in vitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substrate and effector concentrations and possible interactions with other cellular macromolecules, which may modify their kinetic properties. There is a considerable incentive, therefore, to study the properties of enzymes directly in vivo. We have been using non-invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in vivo. We have also developed a novel labelling strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measure the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in vivo.