Introduction of a cysteine protease active site into trypsin

Abstract

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)

Expression in Escherichia coli and Refolding of the Malonyl-/Acetyltransferase Domain of the Multifunctional Animal Fatty Acid Synthase

“…2 It is unlikely therefore that the poor catalytic activity of the Ser-581 3 Cys mutant malonyl-/acetyltransferase results from improper folding of the protein during renaturation in vitro. In this regard the malonyl-/acetyltransferase is typical of most other members of the GXSXG family of hydrolases since replacement of the active site serine residue in subtilisin (19,20), trypsin (21), and acetylcholinesterase (22) also has produced very poor enzymes with k cat values reduced by 2-6 orders of magnitude. In the case of the malonyl-/acetyltransferase it seems likely that replacement of the active site serine with the larger cysteine atom alters the distance between the nucleophile and the catalytic His-683 since the rate of acylation of the nucleophilic residue at position 581 is significantly lowered by the Ser to Cys mutation.…”

Structural and functional diversities in lepidopteran serine proteases

“…2A), which determine (in)sensitivity to PIs. Experiments on synthetic polypeptide enzymes or 'pepzymes' [29] have provided initial clues on the alteration of the activity and/or specificity resulting from modifying key amino acids [30][31][32][33][34][35][36][37]. It does appear that we cannot yet predict functional properties based on structural features.…”

Identification of the Catalytic Residues of Carboxylesterase fromArthrobacter globiformisby Diisopropyl Fluorophosphate-Labeling and Site-Directed Mutagenesis

“…Titration of the sulfhydryl groups of the wild-type and mutant esterases was carried out at 25 C in 100 mM Tris-HCl (pH 8.0) by the method of Ellman.…”

“…The concentrations of released 2-nitro-5-thiobenzoate ions were calculated by the absorption at 412 nm with an extinction coefficient of 13,700 M À1 cm À1 , and the number of SH groups was calculated as described by Higaki et al 25) Other methods. The protein concentrations of the various purified esterases were determined by spectrophotometrically at 280 nm using an extinction coefficient of A280 nm…”