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.

Active site serine 195 of rat anionic trypsin was replaced with a cysteine by site-specific mutagenesis in order to determine if a thiol group could function as the catalytic nucleophile in serine protease active site environment. Two genetically modified rat thiol trypsins were generated; the first variant contained a single substitution of Ser195 with Cys (trypsin S195C) while the second variant contained the Ser195 to Cys as well as an Asp102 to Asn substitution (trypsin D102N,S195C) that more fully mimics the putative catalytic triad of papain. Both variants were expressed as his J signal peptide-trypsin fusion proteins to high levels under the control of the tac promoter. The mature forms of both variants were secreted into the periplasmic space of Escherichia coli. Trypsin S195C shows a low level of activity toward the activated ester substrate Z-Lys-pNP, while both trypsin S195C and trypsin D102N,S195C were active toward the fluorogenic tripeptide substrate Z-GPR-AMC. Esterase and peptidase activities of both thiol trypsin variants were inhibited by known Cys protease inhibitors as well as by specific trypsin inhibitors. The kcat of trypsin S195C was reduced by a factor of 6.4 x 10(5) relative to that of trypsin while the kcat of trypsin D102N,S195C was lowered by a factor of 3.4 x 10(7) with Z-GPR-AMC as substrate. Km values were unaffected. The loss of activity of trypsin D102N,S195C was partially attributed to an inappropriate Asn102-His57 interaction that precludes the formation of the catalytically competent His57-Cys195 ion pair although loss of the negative charge of D102 at the active site probably contributes to diminished activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The eukaryotic serine protease, rat anionic trypsin, and various mutants created by site-directed mutagenesis have been heterologously expressed in Escherichia coli. The bacterial alkaline phosphatase (phoA) promoter was used to control the expression of the enzymes in an induced or constitutive fashion. The DNA coding for the eukaryotic signal peptide of pretrypsinogen was replaced with DNA coding for the phoA signal peptide. The phoA signal peptide successfully directs the secretion of the mammalian trypsinogen to the periplasmic space of E. coli. Active trypsin was expressed in the periplasm of E. coli by deleting the DNA coding for the activation hexapeptide of the zymogen. The activity of trypsin in the periplasm suggests that the enzyme is correctly activated and has folded such that the 12 cysteine residues involved in the six disulfide bonds of rat anionic trypsin have paired correctly. A transcription terminator increased the level of expression by a factor of two. However, increasing the copy number of the plasmid decreased the levels of expression. Localization of the active enzyme in the periplasm allows rapid screening of modified trypsin activities and facilitates the purification of protein to homogeneity and subsequently to crystallinity.

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