16] Affinity labeling of the nucleotide-binding site of myosin light chain kinases

Abstract

This chapter discusses the affinity labeling of catalytic site with specific substrate analogs. Affinity labeling is a technique that can provide information about the nature of crucial residues in the substrate-binding sites of various proteins. The method involves the use of substrate analogs capable of specifically interacting with the protein at the substrate-binding domain and which, in addition, possess a chemically reactive group capable of covalently modifying amino acids that are in close proximity or contribute to that binding site. The ATP analog, p-fluorosulfonylbenzoyl adenosine (FSBA), is an affinity label that has been shown to be an effective reagent for the covalent modification of nucleotide-binding sites of many enzymes and, in particular, several protein kinases. Unlabeled FSBA is used as an inhibitor of myosin light chain kinase (MLSK). To determine the stoichiometry of inactivation, it is necessary to measure the moles of FSBA incorporated into MLSK when the enzyme is inactivated to several different extents.