Experimental Approaches to Studying Enzymes in Vivo: The Application of Nuclear Magnetic Resonance Methods to Genetically Manipulated Organisms

Publisher Summary The view of the intracellular environment is becoming increasingly complex, with a dynamic meshwork of structural proteins and associated enzymes permeating cytoplasmic spaces of water-like viscosity. This structure can have a profound influence on the operation of enzymes in the cell. Understanding of enzymology in the intact cell has grown rapidly in the past twenty years as there have been improvements in the theory of enzyme and pathway kinetics, in the engineering of spectroscopic equipment, and especially in the availability and application of molecular genetic manipulations. One can numerically model the behavior of many complex biological systems, be it the details of catalysis in a single enzyme or the flux in a metabolic pathway, but it seems that experimental approaches to testing the models by studying the cell interior and its biochemistry are somewhat inadequate, although improving rapidly. There are now some more experimental tools to characterize the microenvironment of enzymes in the intact cell and to measure the free metabolite concentrations to which they are exposed. NMR spectroscopy and optical techniques seems to offer complementary approaches, which will allow studies ranging from measurements of subcellular localization in microinjected cells through to whole-organ intact-animal studies. The techniques of molecular genetics are of great value in creating the right experimental models to propel our understanding into the next century.