Preparation and Characterization of the Active Derivative of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) with the Reactive Site Lysine‐15 – Alanine‐16 Hydrolyzed

Jering, Helmut; Tschesche, Harald

doi:10.1111/j.1432-1033.1976.tb10038.x

Cited by 42 publications

(19 citation statements)

References 52 publications

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“…In the absence of the DTT treatment, however, there was no measurable decrease in the concentration of intact protein after 90 min. As expected from previous studies (11,18) Fig. 1.…”

Section: Resultssupporting

confidence: 92%

Genetic dissection of pancreatic trypsin inhibitor.

Goldenberg

Laheru

Wooden

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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In a previous study, a genetic screening procedure was used to identify variants of bovine pancreatic trypsin inhibitor that can fold to an active conformation but that are inactivated much more rapidly than the wild-type protein in the presence of dithiothreitol (DTT). The mechanisms by which 30 of these DTT-sensitive variants are inactivated have now been investigated. Some of the amino acid replacements cause rapid inactivation in the presence of DTT because the three disulfides of the native protein are reduced up to 300-fold faster than for the wild-type protein, leading to complete unfolding. Other substitutions, however, do not greatly increase the rate of complete reduction and unfolding but lead to accumulation of an inactive two-disulfide species. There is a striking correlation between the locations of the DTT-sensitive amino acid replacements in the threedimensional structure of the protein and the mechanisms by which the variants are inactivated. AU of the substitutions that cause rapid unfolding are clustered at one end of the folded protein, in the vicinity of the two disulfides that are reduced most slowly during unfolding of the wild-type protein, while substitutions of the other class are all located at the other end of the protein, near the trypsin binding site. These results indicate that the kinetic stability of native bovine pancreatic trypsin inhibitor and its ability to function as a protease inhibitor are largely influenced by residues in two distinguishable regions of the folded protein.Bovine pancreatic trypsin inhibitor (BPTI) is a small protein, composed of 58 amino acid residues, that has served as an important model for experimental studies of protein folding. The folding process has been analyzed in detail by trapping and characterizing disulfide-bonded intermediates that form when the reduced protein refolds under conditions where folding is coupled to re-formation ofthe three disulfides ofthe native protein (1-4). § In addition, site-directed mutagenesis has been used to test the roles of individual residues in the folding pathway (5-8). As a complement to work with site-directed mutants, we have undertaken a genetic analysis of BPITI using variants isolated by screening randomly mutagenized clones (9 trypsin inhibitor (11). In the screening assay, randomly mutagenized clones were tested to identify those that produce proteins that can fold to an active conformation but are inactivated by DTT much more rapidly than the wild-type protein. The DTT-sensitive amino acid substitutions might cause rapid inactivation by increasing the rate of further reduction and unfolding of IIN or by rendering this species inactive.$ Although the screening assay detects kinetic, rather than thermodynamic, differences among mutant and wild-type proteins, variants with altered inactivation kinetics are likely to display differences in folding thermodynamics as well, since the folding equilibrium is determined by both forward and reverse rates.We have now examined the mechanisms by which these ...

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“…In the absence of the DTT treatment, however, there was no measurable decrease in the concentration of intact protein after 90 min. As expected from previous studies (11,18) Fig. 1.…”

Section: Resultssupporting

confidence: 92%

Genetic dissection of pancreatic trypsin inhibitor.

Goldenberg

Laheru

Wooden

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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“…BPTI with the reactivesite peptide bond (between Lys-15 and Ala-16) cleaved was prepared by using a modification of the procedure in ref. 31, in which 1 of the 3 disulfide bonds of the native protein was selectively reduced, thereby increasing the hydrolysis rate by Ϸ3,000-fold. After treatment with trypsin, the cleaved inhibitor was isolated and the disulfide bond reformed to generate the species designated here as BPTI*.…”

Section: Resultsmentioning

confidence: 99%

Structure of a serine protease poised to resynthesize a peptide bond

Zakharova

Horvath

Goldenberg

2009

Proc. Natl. Acad. Sci. U.S.A.

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The serine proteases are among the most thoroughly studied enzymes, and numerous crystal structures representing the enzymesubstrate complex and intermediates in the hydrolysis reactions have been reported. Some aspects of the catalytic mechanism remain controversial, however, especially the role of conformational changes in the reaction. We describe here a high-resolution (1.46 Å) crystal structure of a complex formed between a cleaved form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site for resynthesis of the peptide bond. This structure defines the positions of the newly generated amino and carboxyl groups following the 2 steps in the hydrolytic reaction. Comparison of this structure with those representing other intermediates in the reaction demonstrates that the residues of the catalytic triad are positioned to promote each step of both the forward and reverse reaction with remarkably little motion and with conservation of hydrogen-bonding interactions. The results also provide insights into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their target proteases.trypsin ͉ bovine pancreatic trypsin inhibitor ͉ enzyme mechanisms S erine proteases are found throughout all 3 domains of cellular life and function in a wide range of physiological processes, including digestion, protein maturation and turnover, hemostasis, and immune responses (1). Approximately 0.6% of human protein-encoding genes are predicted to specify serine proteases, and this family is even more prevalent in other organisms, notably the arthropods (2, 3). A large body of biochemical and structural data have established a 2-step mechanism for hydrolysis of peptide bonds by this class of proteases (4), as shown in Scheme 1.The first step of the reaction is a nucleophilic attack by the catalytic serine residue (Ser-195 in trypsin and other members of the chymotrypsin, or S1, family) on the carbonyl carbon atom of the residue labeled P1, generating a covalent acyl-enzyme intermediate and a new peptide amino terminus, on the P1Ј residue. A second nucleophilic attack, by a water molecule, leads to hydrolysis of the acyl-enzyme, releasing the new carboxyl group and restoring the catalytic Ser residue to its initial state.

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“…No cleavage of this particular bond has so far been detected under conditions which in other classes of proteinase inhibitors lead to a partial proteolytic cleavage [2,3]. However, the modified trypsin-kallikrein inhibitor (Kunitz) could bc prepared in a series of chemical and enzymic reactions [8,10,11]. Modified inhibitor (Kunitz) was inactivated by reaction with carboxypeptidase B.…”

Section: Lysine-15 Repkdcement In Bovine Inhibitormentioning

confidence: 99%

“…Modified trypsin-kallikrein inhibitor (Kunitz) was prepared from virgin inhibitor as already described [8,10,11]. The preparations were tested for homogeneity by polyacrylamide gel electrophoresis and were free from virgin inhibitor when tested for complete inactivation by carboxypeptidase B [8,10,1 I].…”

Section: Modified Inhibitor (Kunitz)mentioning

confidence: 99%

Replacement of Lysine by Arginine, Phenylalanine and Tryptophan in the Reactive Site of the Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) and Change of the Inhibitory Properties

Jering¹,

Tschesche²

1976

European Journal of Biochemistry

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The reactive-site sequence of a proteinase inhibitor can be written as . . . -P,-P2-P,-P;-P;-P;-. . . , where -P,-P;-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikreinm inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue PI, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon.Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys"-inhibitor was reactivated by enzymic replacement of the P, residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme . inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated.The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein@ and plasmin. The homologues with either lysine or arginine in the P, position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg'5-homologue is a slightly more effective kallikrein inhibitor than the Lys"-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys"-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P, residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz). Phe15 and Trp15-inhibitor, virgin trypsin-kallikrein inhibitor homologues with arginine, phenylalanine or tryptophan respectively in place of the Lys-15 reactive-site residue PI ; des-Lys"-inhibitor has the active-site Lys-15 removed by carboxypetidase B. Bz-Arg-Nan, N-bcnzoyl-~~-arginine-4-nitroanilide; Bz-Arg-OEt, N-benzoyl-arginine ethyl ester; Suc-Phe-Nan, N-(3-carboxypropionyl)-phenylalanine-4-nitroanilide; NphGBz, 4-nitrophenyl4'-guanidinobenzoate hydrochloride.

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Preparation and Characterization of the Active Derivative of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) with the Reactive Site Lysine‐15 – Alanine‐16 Hydrolyzed

Cited by 42 publications

References 52 publications

Genetic dissection of pancreatic trypsin inhibitor.

Genetic dissection of pancreatic trypsin inhibitor.

Structure of a serine protease poised to resynthesize a peptide bond

Replacement of Lysine by Arginine, Phenylalanine and Tryptophan in the Reactive Site of the Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) and Change of the Inhibitory Properties

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