[10] Subsite mapping of enzymes: Application to polysaccharide depolymerases

This chapter discusses subsite mapping, the term applied to the experimental determination of the number of subsites, comprising the binding region, the energetics of interaction of each subsite with a monomer residue, and the hydrolytic rate coefficients. The value of the generated subsite map lies in its ability to correctly predict the action pattern of the enzyme. This development is restricted to subsite mapping of polysaccharide depolymerases; however, the general principles developed here are also applicable, within certain limitations, to other polymer-acting enzymes. The interactions of maltotetraose with the binding region of a hypothetical five-subsite amylase are discussed in the chapter. If the substrate binds to expose a susceptible bond to the catalytic amino acids on the enzyme, the complex is productive and bond cleavage can occur. The remaining positional isomers are nonproductive in the sense that they cannot lead to bond scission. The rates of bond scission of the substrate depend on the energetic of binding of the productive positional isomers and on the respective hydrolytic rate coefficients. The chapter explains the best overall procedure for mapping of the binding region of a polysaccharide depolymerase. These are as follows: (1) test for complicating reactions, (2) use bond cleavage frequencies to establish the binding region topography and to measure the apparent subsite binding energies, and (3) use Km and V measured as a function of substrate chain length to complete the subsite map.