The structural determinants of the primary substrate specificity of rat anionic trypsin were examined by using oligonucleotide-directed mutagenesis coupled to a genetic selection. A library was created that encoded trypsins substi- Trypsin is a paradigm for the family of serine proteases that have evolved to cleave peptide bonds after Arg and Lys amino acid residues (9-11). The substrate specificity exhibited by this enzyme family toward these two structurally disparate, positively charged amino acids is defined by the topography of the enzyme, particularly the primary specificity pocket. Crystallographic analysis of trypsin-inhibitor complexes (12-14) and mutagenesis of amino acids that comprise the substrate binding pocket (1,15,16) indicated that two positions, 189 and 190 (trypsin amino acid numbering is based on the chymotrypsinogen amino acid numbering described in ref. 17), are critical in defining the substrate specificity ofthe enzyme. It was anticipated that substitution at these positions would alter function, with a minimum disruption of the structure of the binding pocket, because the remainder of the interactions between the enzyme and the substrate side chain are mediated by main-chain atoms (1).We wished to examine the functional contribution to Arg and Lys specificity of the two amino acids at the base of the substrate binding pocket. A library encoding trypsins substituted at positions 189 and 190 was constructed, and a genetic selection was developed to test the activity of these proteins. A set of mutant trypsins with partially preserved function was isolated and kinetically characterized to investigate the components of Arg and Lys specificity.

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-l-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC and 5'-methylthioadenosine, the committed step in ethylene biosynthesis in plants. Apple

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Aspl02. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Aspl02. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-Nf is solvent inaccessible and is less than 5 A from the catalytic Aspl02. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Aspl02. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Aspl02.Catalysis in the serine proteases is attributed to a triad of amino acids which orchestrate an attack by serine (Ser195 in trypsin) on the substrate carbonyl carbon.

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