25] Application of affinity labeling for studying structure and function of enzymes

Abstract

Publisher Summary This chapter discusses the uses of active-site directed reagents and considers the design and evaluation of such reagents for the benefit of those investigators who may wish to use this approach. In the characterization of an enzyme, it is interesting to try to identify amino acid residues that are at the active site and possibly involved in catalytic activity. An investigator may attempt to design and synthesize a new active-site-directed reagent. The active-sitedirected reagent can be used to determine how environmental factors affect reactivity. Thus the pH dependence of modification can be used to give pK values for the reactive amino acid, or at least for the system of which it is a part. The use of well-designed active-site-directed reagents that closely resemble a substrate, bind tightly to the enzyme, and react with considerable facilitation may also lead one to attempt to position the modifiable residue near a part of the substrate and to assign a catalytic role. In addition, they offer one of the best avenues for the rational development of specific chemotherapeutic agents.