Evolution of Specificity of Protein Proteinase Inhibitors

Laskowski, M.; Kato, Ikuo; Leary, T. Richard; Schrode, James; Sealock, Robert

doi:10.1007/978-3-642-87966-1_66

Cited by 42 publications

(24 citation statements)

References 30 publications

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“…Therefore, the P, specificity of inhibitors has been referred to as the primary specificity [38]. Changes in inhibitory specificity obviously occur in nature by natural mutation of the PI residue.…”

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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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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 resulting two polypeptide fragments must be held together by the adjacent cystine bridges, as is known to be true for other inhibitors [16] .…”

Section: Experimental and Resultsmentioning

confidence: 94%

“…Because from a total of 5.5 ,umol of Bdellin B-3 all sequence studies had to be done, nanomole minus (13)(14)(15)(16)(17)(18)(19)(20)(21) b Cleavage by trypsin (try) or trypsin and chymotrypsin (try/thy) c In stored fractions; in freshly prepared fractions 6 histidine residues were found methods were applied for peptide chromatography [25] , amino acid analysis [26] and sequence studies [27].…”

Section: Experimental and Resultsmentioning

confidence: 99%

Structural homology of a trypsin‐plasmin inhibitor from leeches (bdellin B‐3) with secretory trypsin inhibitors from mammals

Krejci¹,

Fritz²

1976

FEBS Letters

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“…This residue was found invariant in all other structures of this particular protein class that came to our knowledge after the snail inhibitor sequence was completed, i.e. the inhibitor from turtle egg-white [32], Russell's viper venom [33] and the black mamba venom toxins K and I [34] (Fig. 8).…”

Section: Discussionmentioning

confidence: 99%

The Amino‐Acid Sequence of Isoinhibitor K from Snails (Helix pomatia)

Tschesche¹,

Dietl²

1975

European Journal of Biochemistry

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Performic-acid-oxidized isoinhibitor K of snails (Helix pomutiu) was subjected to argininedirected tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg-3 -Pro-4 bond were purified to homogeneity. Four arginine peptides and the C-terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular-ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide < GluGly revealed that the N-terminus was blocked by pyroglutamic acid.The complete amino acid sequence of isoinhibitor K was determined. An almost 50 % homology between the sequences of the snail inhibitor and the bovine trypsin-kallikrein@ inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented. and has a striking similarity in its biochemical properties, its molecular weight and its inhibitory specificity [5,7] to the trypsinkallikrein@ inhibitor (Kunitz) of bovine organs [8]. However, its N-terminus is blocked against Edman degradation. Thus, the amino acid sequence was established by preparing and sequencing of the tryptic peptides after arginine-directed and lysine-directed tryptic cleavages of the performic-acid-oxidized inhibitor. The N-terminus was elucidated by direct mass Abbreviations. < Glu, pyroglutamic acid = pyrrolidone carboxylic acid; Tos-PheCH,CI, l-chloro-4-phenyl-3-tosylamidobutanone; Tos-LysCH,Cl, l-chloro-3-tosylamido-7-aminoheptanone; u, atomic mass units.Enzymes. Trypsin (EC 3.4.21.4); Carboxypeptidase B from porcine pancreas (EC 3.4.12.3); Carboxypeptidase C from orange lcavcs (EC 3.4.12.1).

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Evolution of Specificity of Protein Proteinase Inhibitors

Cited by 42 publications

References 30 publications

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

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

Structural homology of a trypsin‐plasmin inhibitor from leeches (bdellin B‐3) with secretory trypsin inhibitors from mammals

The Amino‐Acid Sequence of Isoinhibitor K from Snails (Helix pomatia)

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