Pressure Effects on the Kinetics of Enzyme-Catalyzed Reactions

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

The effects of high pressure on reaction rates of enzyme-catalyzed reactions will be measured as a function of substrate and inhibitor concentrations. These reactants will be mixed, placed in a steel pressure bomb, and the progress of their reaction will be monitored spectrophotometrically while being subjected to hyperbaric conditions up to 3,000 atmospheres of pressure. Initial velocities of the enzyme-catalyzed reactions will be extracted from the progress curves and fit to original rate equations in order to quantify pressure-sensitive kinetic parameters and to perform model discriminations between reaction mechanisms. The results will be compared to similar effects with isotopically-labeled substrates to determine the effect of pressure on isotope effects. These measured quantities will document the presence, sign and magnitude of volume changes associated with: (1) binding of substrates and inhibitors, (2) rates of chemical transformations including the expression of isotope effects, (3) rates of product dissociation, and (4) rates of protein conformational changes. This is a wholly innovative technological approach to enzymology that will deliver new information regarding the fundamental secret of life: how these modest proteins called enzymes bring billions of orders of magnitude of rate acceleration to the complex biochemical reactions that make up metabolism. For example, this is the first technology to return an exact measurement of a physical parameter associated with a transition-state, namely the activation volume. How activation volumes change with changing conditions will provide a new window of insight into the transition-state chemistry of enzymes. The ultimate goal of this research is to test and challenge the dominant theories of enzymatic catalysis, namely that enzymes accelerate reaction rates by stabilizing transition-states and by binding substrates more tightly in those transition-states.