Clostripain: Characterization of the active site

Biochemistry, 1964

A series of benzosalicylanilide esters has been synthesized and tested for histochemical applicability. Kinetic constants were determined for the hydrolysis of the esters by a-chymotrypsin and rates of hydrolysis were determined for chymotrypsin and trypsin. The specificity evident among the substrates in histochemical studies of chymotrypsinlike enzymes is explicable on the basis of differences in KO. The significance of the uniformly low KO values for these substrates is considered.

Bioscience, Biotechnology, and Biochemistry, 2008

Cathepsin L-like proteinase was purified ~1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH 4 ) 2 SO 4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be ~65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mβNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-β-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mβNA was hydrolyzed maximally by the enzyme (K m value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mβNA, Z-Phe-Val-Arg-4mβNA, Z-Arg-Arg-4mβNA and Z-Ala-Arg-Arg-4mβNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and β-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with K i value 44 × 10 -9 M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg 2+ , Ca 2+ , Cu 2+ , Li 2+ , K + , Cd 2+ , Ni 2+ , Ba 2+ , Mn 2+ , Co 2+ and Sn 2+ also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40ºC. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mβNA was ~50-55ºC with an activation energy E a of ~6.34 KCal mole -1 .

Journal of Agricultural and Food Chemistry, 2000

A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37°C), and exhibited high thermal stability (inactivation required heating for 60 min at 75°C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI) 5.45 and molecular mass) 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value ∼9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-R-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme.The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.

Acta Farmacéutica …, 2002

A new plant endopeptidase has been obtained from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae). Crude extracts were partially purified by organic solvents fractionation: best results (90% of proteins, 94% of total caseinolytic activity and only 9.9% of soluble sugars) were obtained by adding 4 volumes of cold acetone to the crude extract. This preparation (redissolved acetone precipitate, RAP) showed maximum activity (> 80%) at pH 7.3-10.7, and exhibited high thermal stability (80% of residual activity after heating for 30 min at 60 ºC). Low sodium chloride concentrations (0.2 M) does not affect caseinolytic activity, but diminishes with the increase of salt concentration (52% of residual activity at 2.5 M NaCl). RAP showed also clotting activity and a notably preference for κ κ casein over α α s1 , α αs 2 and β β caseins, properties that could be of interest in the cheese industry. The enzyme was completely inhibited by E-64 and iodoacetic and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group, as all the other studied proteases belonging to the family Bromeliaceae. IEF-zymogram of RAP showed six bands (pI 5.9 to >9.3), most of them proteolytically actives, but only three of which (pI 6.4, 8.3 and >9.3) proved to be important. Cation exchange chromatography (FPLC) allowed the isolation of the main fraction, named hieronymain I (pI = >9.3, molecular weight = 25 kDa). RESUMEN. "Purificación de una nueva endopeptidasa aislada de frutos de Bromelia hieronymi Mez (Bromeliaceae)". Se ha aislado una nueva endopeptidasa a partir de frutos inmaduros de Bromelia hieronymi Mez (Bromeliaceae). Los extractos crudos fueron parcialmente purificados por fraccionamiento con solventes orgánicos, habiéndose obtenido los mejores resultados al precipitar el extracto crudo con 4 volúmenes de acetona fría (el precipitado retiene el 90% de las proteínas y el 94% de la actividad caseinolítica y contiene sólo 9,9% de azúcares solubles). El precipitado acetónico redisuelto (RAP) mostró máxima actividad (> 80%) a pH 7,3-10,7 y muestra gran estabilidad térmica (mantiene 80% de actividad residual luego de calentarlo a 60 ºC durante 30 min). Bajas concentraciones de cloruro de sodio (0,2 M) no afectan la actividad caseinolítica, pero ésta disminuye con el incremento de concentración salina (52% de actividad residual cuando la concentración de cloruro de sodio es 2,5 M). El RAP demostró poseer actividad coagulante de la leche y notable preferencia por la fracción κ caseína por encima de las α s1 , α s2 y β caseínas, características que pueden resultar de interés en la industria quesera. La enzima resultó totalmente inhibida por E-64 y ácido iodoacético y activada por cisteína, lo que sugiere que pertenece al grupo de la endopeptidasas cisteínicas, en el que se encuentran todas las proteasas estudiadas de la familia Bromeliaceae. El isoelectroenfoque seguido de zimograma revela la presencia de seis bandas (pI 5,9 a > 9,3), la mayoría de ellas proteolíticamente activas, pero sólo tres (pI 6,4, 8,3 y >9,3) de importancia. Por cromatografía de intercambio catiónico (FPLC) se logró aislar la fracción principal, denominada hieronymina I (pI >9,3 peso molecular 25 kDa).

Journal of Agricultural and Food Chemistry, 2000

A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37°C), and exhibited high thermal stability (inactivation required heating for 60 min at 75°C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI) 5.45 and molecular mass) 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value ∼9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-R-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme.The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.

FEBS Letters, 1992

A prolyl cndopcptidase (PEPasc, EC X4.21.26) that specifictally clcavcs 1hc IS-kDa protein ofphotosystcm ii was cxtracthti from photosystem II mcmbrancs with 1 M NaCI. Protcolytic activity mcasurcd with anificial substrates was ICW 1han u quarter of that with the protein. Studies on ~nhibitiou of the protcolysis by an artificial substrate suggested lhat the protestsc rceognizcs the scissi~c prolyt bond. The protcasc aas inhibited by CuCl?. but not by diisopropyl fluorophosphdlc or p-chloromcrcuriphmylsulforGc acid. Thcsc findings suggest that the protea= rcprc=ts a new &ass of PEPaxc. The specificity of the cnzymc is discussed in relation 10 the structure of 1h1: IX-k& protein.

FEBS Letters, 1976

Archives of Biochemistry and Biophysics, 2006

Cystalysin, the key virulence factor in the bacterium Treponema denticola responsible for periodontis, is a pyridoxal 5 0 -phosphate (PLP) enzyme which catalyzes, in addition to a,b-elimination of L-cysteine, racemization and transamination of both enantiomers of alanine. In this paper several indicators have been used as probes of the different conformational status of T. denticola cystalysin in the holo and apo form. Compared to holoenzyme, the apoenzyme displays an altered reactivity of cysteine residues, a significantly different pI, and a differential susceptibility to proteinase K. The site of cleavage that is accessible in apocystalysin and masked in holocystalysin has been identified by mass spectrometry as the peptide bond between Phe 360 and Gly 361. This cleavage results in the loss of the C-terminal fragment corresponding to a molecular mass of 4289.21 ± 0.1 Da. The major fragment of cleaved enzyme retains its dimeric structure, binds the coenzyme with an affinity $5000-fold lower than that of uncleaved holoenzyme, and in the reconstituted form is able to form the external aldimine with substrates. Although the break causes the loss of lyase, racemase and transaminase activities of D-alanine, it does not abolish the transaminase activity of L-alanine. Possible mechanistic and physiological implications are proposed. (C.B. Voltattorni).

FEBS Journal, 2005

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in a-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 ± 0.3 • 10)11 m and thus works as a highly potent competitive inhibitor. The commercially available a-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated a-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mm sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 m ammonium sulfate. The rectangular crystals of small dimensions of 0.24 • 0.15 • 0.10 mm 3 were obtained. The X-ray intensity data were collected at 2.2 Å resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of a-chymotrypsin, which was readily interpreted as a 14 residue fragment of a-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 r between the active site Ser195 Oc and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 Å between Ser195 Oc and Trp29 C

Febs Journal, 2004

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in α-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 ± 0.3 × 10−11 m and thus works as a highly potent competitive inhibitor. The commercially available α-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated α-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mm sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 m ammonium sulfate. The rectangular crystals of small dimensions of 0.24 × 0.15 × 0.10 mm3 were obtained. The X-ray intensity data were collected at 2.2 Å resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of α-chymotrypsin, which was readily interpreted as a 14 residue fragment of α-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16–29). The electron density for the eight residues of the C-terminus, i.e. Ala22–Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16–Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 σ between the active site Ser195 Oγ and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 Å between Ser195 Oγ and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16–Trp29. In addition to the O–C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of α-chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of α-chymotrypsin.