Evolutionary Divergence of Substrate Specificity within the Chymotrypsin-like Serine Protease Fold*

One of the enduring paradigms in enzymology is the theme of evolutionary divergence in substrate specificity from a parental enzyme possessing a prototypical fold. Within the fold of each divergent enzyme is embedded the catalytic machinery essential to providing rate enhancement for an identical reaction, and the particular amino acids directly involved in this function are expected to be highly conserved and invariant in their spatial positions. Among the remaining residues will be those required for the overall three-dimensional structure. Such residues are found largely within the hydrophobic core of the enzyme and, within a family, may show covariance according to the thermodynamic determinants specifying stability and the informational determinants specifying uniqueness of the fold. The remaining amino acids of the enzymes then constitute much of the raw material for evolutionary adaptation to novel selectivity. Many of these can be expected to be found interacting with distal, variable, specificitydetermining portions of the substrates in modes that are unique to each enzyme of the family. The core structural residues may also contribute to generating new specificity, by virtue of second or third shell interactions with the amino acids directly contacting substrate. Here we describe structure-function relationships in one of the largest and most comprehensively studied of all enzyme families, the serine proteases with a chymotrypsin fold. Over 20 unique structures have been determined to date, and the number of available sequences exceeds 500 (for a comprehensive review, see Ref. 2). All of the enzymes possess an identical fold consisting of two b-barrels, with the catalytic Ser, His, and Asp amino acids found at the interface of the two domains. Common features present in all structures, including five enzyme-substrate hydrogen bonds at positions P1 and P3, serve to properly juxtapose the scissile peptide bond adjacent to the Ser-His catalytic couple, such that the nucleophilic Ser O-g is accurately positioned for attack. Given these conserved interactions in the direct vicinity of the reacting groups, the more distal contacts sharply diverge. We describe the structural themes that embody this divergence together with some of the most important enzymological data necessary to the structure-function correlation. One important theme that emerges is that of catalytic register (3); the divergence in distal interactions in different enzymes must be such as to still permit accurate substrate alignment. Another observation is that many structural determinants controlling specificity reside on surface loops, and this allows for the possibility of rapid and varied evolutionary divergence with conservation of the overall tertiary fold. Divergence of substrate specificity within the context of a common structural framework represents only one mechanism by which nature is able to evolve new activities. Another mechanism involves incorporation of new catalytic groups within an active site, such that the same scaffold can carry out a range of chemistries with just a single step common to different members of the family. This theme is elaborated in the review by Babbitt and Gerlt (4). It is also possible to obtain information on the requirements for a common catalytic function by studying examples of convergent evolution, where the same chemical reaction is carried out by very different scaffolds. Serine proteases represent a paradigm in this respect as well; besides the chymotrypsin-fold enzymes, there are now four other known natural folds that possess the requisite catalytic determinants in similar spatial positions (5–10). Study of these structures and redesigned versions of trypsin (11) shows that the catalytic Asp can adopt virtually any position with respect to the Ser-His dyad, suggesting that the classical “catalytic triad” of Ser, His, and Asp residues (Fig. 1) can in fact be better described as the juxtaposition of two dyads: Ser-His and His-Asp (6).