抄録
The outline of the subsite theory was described which correlates the action patterns of amylases, i, e., the dependency of rate parameters on the degree of polymerization n of linea rsubstrates and the mode of cleavage of maltooligosaccharides, with the subsite structure of the enzyme. The Subsite structure is defined as the arrangement of a definite number of subsites constituting the active site of an amylase, each of which interacts specifically with a glucose residue of linear substrates with its own "subsite affinity" designated AZ (in units of kcal/mole). An n-mer substrate can be bound with the active site in a variety of "productive" and "nonproductive" binding modes, and the probability of a particular binding mode is determined by the subsite structure of the enzyme . Assuming that the subsite affinities are additive and that the intrinsic rate constant kint of substrate bond cleavage in a productive complex is independent of n and the binding mode, the action pattern of an amylase can quantitatively be described in terms of Ai's and kint ( the properties of the subsite structure), and vice versa. Subsite structures of four kinds of amylase, glucoamylase, Taka-amylase A, bacterial liquefying a-amylase and wheat bran j3-amylase, which have been evaluated from the ndependency of rate parameters, were displayed in histograms. For the three amylases other than glucoamylase, negative value of Subsite affinity is commonly seen at a subsite adjacent to the catalytic site, suggeting the "strain" in the enzyme-substrate complex . This explains the reason why maltose is hardly hydrolyzed by these enzymes . It was demonstrated that the subsite structure of glucoamylase can reasonably account fo rits substrate specificity towards synthetic substrates, i, e., the unexpectedly lower molecular activity for phenyl a-gulcoside compared with that for phenyl a -maltoside . The subsite theory is also useful to predict or interprete the change in action patte rns of amylases caused by altering one of the subsite affinities by chemical modificatio n. Calculation with Taka-amylase A showed that the molecular activity towards maltose could increase by afactor of several tens whereas that towards maltoheptaose decreases five fold, if a particular subsite affinity is decreased by 3 kcal/mole . An example of photooxidation of a histidine residue of bacterial liquefying a-amylase was described, in which the modified subsite was located by studying the effect of modification as a function of n . The feasibility of changing artificially the action patterns of amylases has thus been substantiated.
