19] Characterization of enzyme-complex formation by analysis of nuclear magnetic resonance line shapes

Abstract

This chapter discusses the characterization of enzyme–complex formation by analysis of nuclear magnetic resonance (NMR) line shapes. A general approach involving the analysis of NMR line shapes for obtaining chemical exchange rates is described here. The analysis requires measuring changes in line widths and peak positions as a function of ligand concentration from a simple one-dimensional spectrum. The chapter illustrates the description of the general approach with a simple kinetic scheme, followed by the description of a specific example using results from 31 P NMR of the enzyme phosphoglucomutase. A cyclic scheme for binding of a substrate analog to the mono- and di-Cd 2+ complexes of the phospho form of the enzyme is readily characterized with this method. In this system, two magnetic nuclei undergo exchange because of binding—namely, the 31 P nucleus of the ligand and that of the phosphoenzyme. Hence, additional constraints are involved; the spectral characteristics of two sets of exchange sites must be consistent with the same association–dissociation rate constants.