Structural origins of substrate discrimination in trypsin and chymotrypsin

Abstract

Converting the specificity of trypsin to that of chymotrypsin has been shown to require the exchange of amino acids in multiple portions of the protein, including two surface loops which do not directly contact the substrate. Crystallographic analysis of two mutant trypsins possessing chymotrypsin-like specificity now reveals that these distal surface loops alter function by directly determining the structure of the primary binding site. Efficient acylation of cognate substrates correlates with a distinct backbone conformation of the conserved Gly216 residue. This amino acid is located on the surface of the specificity pocket and forms two main-chain hydrogen bonds with a nonspecific portion of substrate. By contrast, the improvement in substrate binding affinity effect by the substitution of the distal Tyr172 residue with Trp derives from structural rearrangements at the extreme base of the pocket. Together, the kinetic and crystallographic data strongly suggest that both Asp189 and Gly216 must be considered as primary determinants of substrate specificity in trypsin.