Substrate analogs, enzymes, conformationally restricted

Probing the Active Sites of Enzymes with Conformationally Restricted Substrate Analogs. BY G. L. KENYON AND J. A. FEE, Department of Chemistry, University of California, Berkeley, California. 381... 

Substrate analogs which promise to be particularly good active-site probes are those which are conformationally restricted. One key feature of enzymatic processes is that when a substrate is bound to an enzyme, probably only one of the many possible conformations of the substrate molecule is assumed. Consequently, before a detailed mechanism for an enzymatic process can be formulated, the preferred conformations of each of the enzyme-bound substrates must be known. ... 

Besides this problem of designing conformationally restricted analogs for highly specific enzymes, there are Other problems to be considered in dealing with less specific enzymes. These are discussed later in the section on locked a-chymotrypsin substrates. 

Use of conformationally restricted substrate analogs for investigating the substrate specificity of a-chymotrypsin provides an instructive example of the difficulties encountered in interpreting the results of such experiments, difficulties which, as we shall see, are especially severe for relatively nonspecific enzymes. 

In conclusion, one must be aware of these limitations on the use of locked substrate analogs. The problems encountered in the study of a-chymotrypsin are perhaps more severe than for most other enzymes, since a-chymotrypsin normally acts on large, polymeric substrates and is relatively nonspecific. The active site of a-chymotrypsin therefore potentially can bind small substrates such as D24 in a variety of ways. Ideally, larger conformationally restricted substrates should give more information about the active site of a-chymotrypsin. However, besides the increased problems involved in synthesizing these larger substrates, there is the problem of increased possibility of uncertainty in their conformations. 

Conformationally restricted analogs of substrates can be useful in elucidating both the substrate specificities and the product specificities of enzymes. The restriction can help stabilize an intermediate in the enzymatic process so that it may be isolated. Two or more otherwise structurally equivalent portions of a substrate may be rendered nonequivalent by the restriction so that potential differentiation of these portions by the enzyme in determining product specificity may be investigated. 

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