Spectroscopic Quantitation of H-bond Strength in Enzyme-Substrate Complexes

Case Western Reserve University, Cleveland OH

Investigators

Abstract

The presence of a single H-bond at an enzyme active site can be crucial to catalysis. Site directed mutagenesis has revealed that removing an H-bond donor or acceptor can reduce an enzyme's catalytic efficiency by a factor of as much as 105. The researchers are developing two novel methods of determining both the strength and direction of individual H-bonds between a substrate or inhibitor and functional groups in the enzyme active sites. These two methods are based on the use of stable isotopes. Preliminary results have shown that the C-D stretching frequency of a primary or secondary alcohol depends directly on the strength of the H-bond from the alcohol. This change in frequency also results in equilibrium isotope effects on the formation of the complex and kinetic isotope effects that can be used to characterize the transition state of the reaction. As an example, the H-bond in cyclic-AMP dependent protein kinase between the aspartate residue that may serve as a critical H-bond acceptor and the nucleophilic serine will be characterized. X-ray crystallography suggests that the aspartate is in the correct position to activate the hydroxyl group of a serine residue for its role as the nucleophile in an ATP-dependent phosphoryl transfer reaction. In spite of the crystallographic proximity of the aspartate, its role in catalyzing the reaction has been controversial. The incorporation of the present studies into the chemical mechanism of cAMP-dependent protein kinase will provide a paradigm for this large family of enzymes involved in signal transduction. Understanding the mechanism of protein kinases will contribute to our ability to design and manipulate these important enzymes that provide for the immediate control of many biological functions. The unique insight into the activation of the nucleophile of an ATP-dependent phosphoryl transfer will extend the current understanding of all kinase reactions.