Bovine Chymotrypsinogen Research Articles

The role of amino-terminal alanine in the control of conformation and activity of alpha-chymotrypsin.

Novel acetylated derivatives of three different three-chained chymotrypsins were prepared from bovine chymotrypsinogen A and their catalytic properties and kinetics of denaturation in urea were compared with those of the corresponding non-acetylated enzymes. Measurements of the Km (apparent) as a function of pH confirmed earlier findings of Valenzuela and Bender [J. Biol. Chem. 248, 4909-4914 (1973)] that the alpha-species of chymotrypsin is much more sensitive to reversible inactivation at high pH compared to its sister three-chained chymotrypsins, alpha 1 and kappa-chymotrypsin. Similarly, the denaturation rate constants in 8 M urea of alpha-chymotrypsin were much more sensitive to high pH than alpha 1 and kappa-chymotrypsin. The urea denaturation study showed a transition at about pH 8 to a more urea-sensitive form of alpha-chymotrypsin, whereas alpha 1 and kappa-chymotrypsin were relatively insensitive to a change in pH from 6.5 to 10. When the N-terminal Ala149 of alpha-chymotrypsin was acetylated, thus preventing protonation of the N terminus, the active enzyme derivative displayed the same Km (app) vs pH profile as alpha 1 and kappa-chymotrypsin. Urea denaturation studies with this masked derivative also showed that the pH-dependent transition of native alpha-chymotrypsin at pH 8 was eliminated. These results demonstrate that it is the presence of the protonated Ala149 residue in alpha-chymotrypsin that accounts for much of the hypersensitivity of this enzyme species to inactivation and urea denaturation in the pH region 7.5-10.

Further studies on subunit III of bovine procarboxypeptidase A: Structure and reactivity of the weakly functional active site

FEBS LettersVolume 128, Issue 1 p. 13-16 Full-length articleFree Access Further studies on subunit III of bovine procarboxypeptidase A Structure and reactivity of the weakly functional active site Catherine Wicker, Catherine Wicker Centre de Biochimie et de Biologie Moléculaire du CNRS, 31, Chemin Joseph-Aiguier, 13274 Marseille CEDEX 2, FranceSearch for more papers by this authorAntoine Puigserver, Antoine Puigserver Centre de Biochimie et de Biologie Moléculaire du CNRS, 31, Chemin Joseph-Aiguier, 13274 Marseille CEDEX 2, FranceSearch for more papers by this author Catherine Wicker, Catherine Wicker Centre de Biochimie et de Biologie Moléculaire du CNRS, 31, Chemin Joseph-Aiguier, 13274 Marseille CEDEX 2, FranceSearch for more papers by this authorAntoine Puigserver, Antoine Puigserver Centre de Biochimie et de Biologie Moléculaire du CNRS, 31, Chemin Joseph-Aiguier, 13274 Marseille CEDEX 2, FranceSearch for more papers by this author First published: June 01, 1981 https://doi.org/10.1016/0014-5793(81)81067-8Citations: 13AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume128, Issue1June 01, 1981Pages 13-16 ReferencesRelatedInformation

Interaction of chymotrypsinogens with alpha 1-protease inhibitor.

In a previous report [Largman, C., Brodrick, J.W., Geokas, M.C., Sischo, W.M., & Johnson, J.H. (1979) J. Biol. Chem. 254, 8516-8523] it was demonstrated that human proelastase 2 and alpha 1-protease inhibitor react slowly to form a complex that is stable to denaturation with sodium dodecyl sulfate and beta-mercaptoethanol and that the zymogen can be recovered from the isolated complex following dissociation by hydroxylamine. The present report demonstrates that bovine chymotrypsinogen A reacts with human alpha 1-protease inhibitor in a very similar manner. The rate of complex formation was measured by two methods. In the first, the reaction was followed by determining the loss of the inhibitory activity of alpha 1-protease inhibitor as a function of time. A second-order rate constant for complex formation formation (pH 7.6, 36 degrees C) of 12.9 +/- 2.4 M-1s-1 was obtained. In the second procedure, the reaction of fluorescein isothiocyanate labeled chymotrypsinogen A with alpha 1-protease inhibitor was measured by fluorescence polarization. A second-order rate constant (pH 7.6, 37 degrees C) of 13.9 +/- 2.1 M-1s-1 was obtained. The rate of complex formation is approximately 10(-5) of that measured for the reaction of bovine chymotrypsin with alpha 1-protease inhibitor. Dissociation of the complex was not observed after dilution or the addition of excess bovine alpha-chymotrypsin. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments, human chymotrypsinogens I and II react with alpha 1-protease inhibitor at rates that are approximatley equivalent to that determined for bovine chymotrypsinogen A. In contrast, bovine trypsinogen reacts very slowly with alpha 1-protease inhibitor, at a rate that is at most 10(-2) of that of bovine chymotrypsinogen A. These results suggest that zymogens react with alpha 1-protease inhibitor by virtue of partially formed active sites and that the potential active-site specificity of the zymogen in part determines the rate of complex formation.

Transpancreatic transport of digestive enzyme

When porcine α-amylase or bovine chymotrypsinogen A was added to the medium bathing the rabbit pancreas in short-term organ culture, the secretion of these enzymes collected via the duct system increased greatly. To determine if it was indeed the amylase added to the bath that was recovered in secretion, endogenous enzyme stores were prelabeled during a 4 h incubation with [ 3H]- leucine and the specific radioactivity of amylase in secretion followed. The addition of unlabeled exogenous amylase to the bathing medium reduced the specific radioactivity of secreted amylase of about 90% suggesting that the response was due to the transpancreatic transport of the added enzyme. This inhibition was maintained over time, and was a result, not only of the increased secretion of unlabeled enzyme, but also of a 72% steady-state inhibition in the secretion of endogenous (labeled) amylase. This latter observation indicates that the exogenous enzyme crosses the acinar cell and mixes with endogenous cellular stores. A cellular route is also suggested by the observation that the addition of amylase to the bath increased the amylase concentration in ductal fluid relative to that in the bath by about 20 times; it did not reduce it as would be expected if paracellular shunts were involved. In addition, a cellular pathway is suggested by the observation that a 2 h prior incubation in bovine chymotrypsinogen resulted in a greatly augmented chymotrpsinogen response to a maximal cholinergic stimulus. In all, the data support the prediction of the equilibrium theory of digestive enzyme secretion that enzyme secretion should be responsive to mass action, and the prediction of the enteropancreatic circulation hypothesis that a capacity exists for a substantial transpancreatic flux of digestive enzyme.

Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha.

Reversible unfolding of bovine chymotrypsinogen A in 2H2O either by heating at low pH or by exposure to 6 M guanidinium chloride results in the exchange of virtually all the nitrogen-bound hydrogens that give rise to low-field 1H NMR peaks, without significant exchange of the histidyl ring Cepsilon1 hydrogens. These preexchange procedures have enabled the resolution of two peaks, using 250-MHz correlation 1H NMR spectroscopy, that are attributed to the two histidyl residues of chymotrypsinogen A. Assignments of the Cepsilon1 hydrogen peaks to histidine-40 and -57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidyl Cepsilon1 H peaks were also resolved with solutions of preexchanged chymotrypsin Aalpha. The histidyl peaks of chymotrypsin Aalpha were assigned by comparison of NMR titration curves of the free enzyme with those of its complex with bovine pancreatic trypsin inhibitor (Kunitz). The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit two inflections; the additional inflections were assigned to interactions with neighboring carboxyl groups: aspartate-102 in the case of histidine-57 and aspartate-194 in the case of histidine-40 of the zymogen. In bovine chymotrypsinogen A in 2H2O at 31 degrees C, histidine-57 has a pK' of 7.3 and aspartate-102 a pK' of 1.4, and the histidine-40-aspartate-194 system exhibits inflections at pH 4.6 and 2.3. In bovine chymotrypsin Aalpha under the same conditions, the histidine-57-aspartate-102 system has pK' values of 6.1 and 2.8, and histidine-40 has a pK' of 7.2. The results suggest that the pK' of histidine-57 is higher than the pK' of aspartate-102 in both zymogen and enzyme. A significant difference exists in the structure and properties of the catalytic center between the zymogen and activated enzyme. In addition to the difference in pK' values, the chemical shift of histidine-57, which is highly abnormal in the zymogen (deshielded by 0.6 ppm), becomes normalized upon activation. These changes may explain part of the increase in the catalytic activity upon activation. The 1H NMR chemical shift of the Cepsilon1 H of histidine-57 in the chymotrypsin Aalpha-pancreatic trypsin inhibitor (Kunitz) complex is constant between pH 3 and 9 at a value similar to that of histidine-57 in the porcine trypsin-pancreatic trypsin inhibitor complex [Markley, J.L., and Porubcan, M. A. (1976), J. Mol. Biol. 102, 487--509], suggesting that the mechanisms of interaction are similar in the two complexes.

Ionic interactions between bovine chymotrypsinogen A and chondroitin sulfate A.B.C.. A possible model for molecular aggregation in zymogen granules.

The formation of large aggregates by ionic interactions between acidic glucosaminoglycans and cationic secretory proteins has been proposed as one of the critical steps in the concentration process in the condensing vacuoles of secretory cells. In this paper, this hypothesis was tested by studies on the interactions between bovine chymotrypsinogen A and chondroitin sulfate as a simplified model. Small amounts of chondroitin sulfate were found able to induce chymotrypsinogen precipitation. Like zymogen granules, the resulting aggregates were moderately sensitive to ionic strength and insensitive to osmolality. Moreover, their pH dependence was similar to that of isolated zymogen granules. When sulfated glucosaminoglycans isolated from the zymogen granules of the guinea pig pancreas were used instead of chondroitin sulfate, the same kind of interactions with chymotrypsinogen were obtained. Our data support the hypothesis that the strong ionic interactions between those sulfated glucosaminoglycans and cationic proteins could be responsible for the concentration process.

On the activation of bovine chymotrypsinogen A. Conformational isomerization of alpha1- and kappa-chymotrypsin and their autolytic conversion to alpha- and gamma-chymotrypsin.

On the activation of bovine chymotrypsinogen A. Conformational isomerization of alpha1- and kappa-chymotrypsin and their autolytic conversion to alpha- and gamma-chymotrypsin.

Further characterization of subunit III of bovine procarboxypeptidase A-S6 as a non activatable zymogen

The homology of Subunit III of bovine procarboxypeptidase A-S6 (EC 3.4.12.-) with Subunit II (bovine chymotrypsinogen C) of the same complex, already reported in a previous publication (Puigserver, A. and Desnuelle, P. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2442–2445) has been extended to the position of the single methionine of the chains. The sequences linked by 4 disulfide bridges out of 5 are also probably homologous in the 2 proteins. The last bridge is displaced in Subunit III as a consequence of the deletion of the N-terminal half-cystine. Subunit III, which is not activatable by trypsin, due to the loss of essential residues in the N-terminal region, has conserved a weakly functional active site reacting with concentrated diisopropylfluorophosphate at exactly the same rate as that of Subunit II.

Estimation of tryptophyl and tyrosyl exposure in tryptophan-rich proteins by ultraviolet difference spectrophotometry. Lysozyme and Chymotrypsinogen.

Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophanamide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (delta epsilon) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287 nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because delta epsilon285 was larger than delta epsilon292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins. The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more beta-structured for chymotrypsinogen.

The charge-relay system of serine proteinases: Proton magnetic resonance titration studies of the four histidines of porcine trypsin

Peaks corresponding to the C (2)-protons of all four histidine residues of porcine β-trypsin were resolved in 250 MHz nuclear magnetic resonance spectra after deuteration of the slowly exchangeable N- H groups (whose resonances obscure the histidine peaks) by reversible unfolding of the protein in D 2O. One of the four peaks was assigned to the charge-relay histidine in the active site of trypsin (His(57) in the bovine chymotrypsinogen numbering system). Whereas the three other histidine C (2)-peaks exhibited normal titration curves with single p K′ values of 7.20, 6.71 and 6.67, the peak assigned to His(57) had an abnormal titration curve showing two protonation steps in the pH range from 1 to 9. The first protonation with a pH′ mid of 5.0 is rapid on the nuclear magnetic resonance time-scale; the second with a pH′ mid of 4.5 is slow and apparently involves conformational transitions between two states having lifetimes of approximately 18 ms. In the complex between porcine β-trypsin and bovine pancreatic trypsin inhibitor (Kunitz) His(57) was found to be insensitive to pH over the range from 4 to 9 and its chemical shift resembles that of His(57) in the singly protonated charge relay of free trypsin. This result provides direct evidence that the trypsin charge relay acts as a proton acceptor in the initial catalytic step which leads to the formation of a tetrahedral complex. In the presence of equimolar bovine pancreatic trypsin inhibitor (Kunitz) the pH' mid of the conformational transition that affects the charge-relay histidine is lowered from 4.5 to approximately 3.5.

Dissociation of bovine 6S procarboxypeptidase A by reversible condensation with 2,3-dimethyl maleic anhydride: application to the partial characterization of subunit III.

Bovine 6S procarboxypeptidase A can be dissociated into its three subunits by acylation with dimethyl maleic anhydride. The deacylated subunits are obtained in a largely native form due to instability of the bonds to dimethyl maleate at pH values near neutrality. The seven first residues of subunit III are identical to residues 18-24 of bovine chymotrypsinogen B and very similar with the same residues in bovine chymotrypsinogen A and C (subunit II) and also in proelastase A of the African lungfish. Therefore, this subunit is likely to be a chymotrypsinogen or proelastase-A-like zymogen which has lost the ability to be activated on account of a deletion of the N-terminal residues from half-cystine 1 to valine 17. Like other pancreatic zymogens, subunit III appears to possess a weakly functional active site.

Aspergillus oryzae acid proteinase. Purification and properties, and formation of π-chymotrypsin

An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.

Transport of bovine chymotrypsinogen into rabbit pancreatic cells.

ARTICLESTransport of bovine chymotrypsinogen into rabbit pancreatic cellsC Liebow, and SS RothmanC Liebow, and SS RothmanPublished Online:01 May 1974https://doi.org/10.1152/ajplegacy.1974.226.5.1077MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByThe incoherence of the vesicle theory of protein secretionJournal of Theoretical Biology, Vol. 245, No. 1Conservation of Digestive EnzymesStephen Rothman, Charles Liebow, and Lois Isenman1 January 2002 | Physiological Reviews, Vol. 82, No. 1Membrane protein transport in eukaryotic secretion cellsTransport of proteins across membranes — a paradigm in transitionBiochimica et Biophysica Acta (BBA) - Reviews on Biomembranes, Vol. 1241, No. 3A trans-membrane pore can account for protein movement across zymogen granule membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1238, No. 1The effect of ethanol on enzyme synthesis and secretion in isolated rat pancreatic lobulesBiochemical Pharmacology, Vol. 36, No. 20The ‘Endocrine’ Enzyme Secretion from the Pancreas8 July 2009 | Scandinavian Journal of Gastroenterology, Vol. 21, No. 5Origin of the decline in pancreatic amylase secretion with time in vitroJ. J. Ho, and S. S. Rothman1 July 1983 | American Journal of Physiology-Cell Physiology, Vol. 245, No. 1Gastroenterology, Vol. 85, No. 6Exogenous secretory proteins do not inhibit carbamylcholine-stimulated release of pulse-labelled secretory proteins from rat pancreatic tissue slicesBiochimica et Biophysica Acta (BBA) - General Subjects, Vol. 632, No. 1Large decrease in zymogen granule size in the postnatal rat pancreasJournal of Ultrastructure Research, Vol. 70, No. 2Transpancreatic transport of digestive enzymeBiochimica et Biophysica Acta (BBA) - General Subjects, Vol. 585, No. 3Amylase transport across ileal epithelium in vitroBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 512, No. 1Chymotrypsinogen · inositol phosphatide complexes and the transport of digestive enzyme across membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 509, No. 2SYNTHESIS, INTRACELLULAR TRANSPORT AND DISCHARGE OF EXPORTABLE PROTEINS IN THE PANCREATIC ACINAR CELL AND OTHER CELLSBiological Reviews, Vol. 53, No. 2La Sécrétion Des Enzymes Digestifs Chez Les Vertébrés: Part 126 September 2008 | Archives Internationales de Physiologie et de Biochimie, Vol. 85, No. 4La Sécrétion Des Enzymes Digestifs Chez Les Vertébrés: Part 226 September 2008 | Archives Internationales de Physiologie et de Biochimie, Vol. 85, No. 4Equilibration of pancreatic digestive enzymes across zymogen granule membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 455, No. 1Protein Transport by the PancreasScience, Vol. 190, No. 4216Enteropancreatic Circulation of Digestive EnzymesScience, Vol. 189, No. 4201 More from this issue > Volume 226Issue 5May 1974Pages 1077-1081 Copyright & PermissionsCopyright © 1974 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1974.226.5.1077PubMed4824859History Published online 1 May 1974 Published in print 1 May 1974 Metrics

46] Chymotrypsin(s)

Chymotrypsins are the products of the tryptic activation of the inactive precursors, the chymotrypsinogens produced by the pancreas of many species. The isolation of bovine chymotrypsinogen and its activation by trypsin were reported some forty years ago. The initial step is the splitting of the Arg 15 -He 16 peptide bond leading to the formation of catalytically active π-chymotrypsin. Subsequent autocatalytic events give rise to δ-, K-, and γ,-chymotrypsins as intermediates in the formation of α-chymotrypsin. The last-mentioned species, being more stable than its precursors, is the one most commonly encountered. All active forms of the enzyme are known to exhibit a marked preference in catalyzing the hydrolysis of peptide bonds on the carboxyl side of aromatic amino acid residues. While the procedures for the isolation of chymotrypsin and chymotrypsinogen in a highly homogeneous state have been well developed, the preparation of chymotrypsinogen totally devoid of chymotrypsin or of the latter free of zymogen and other enzymes normally found in the starting material is often difficult to achieve, even after repeated purification procedures. Similar considerations may apply during studies on the effects of chemical modification of the protein on the enzymatic activity, where it is often desirable to separate active from inactive species.

The coupling of chymotrypsinogen to bovine albumin

Bovine chymotrypsinogen has been couple to bovine albunin and the resulting complex has been to retain enzymatic and immunological properties.

Influence of the substitution of tryptophan 215 in bovine chymotrypsinogen A on its potential enzymic activity

Influence of the substitution of tryptophan 215 in bovine chymotrypsinogen A on its potential enzymic activity

AIR-Reoxidation of reduced, denatured chymotrypsinogen A covalently attached to a solid matrix

Bovine chymotrypsinogen A was covalently attached to porous succinylglass beads via the zymogen's amino groups. The bound zymogen was completely reduced in 8 M urea, then allowed to reoxidize, and activated to chymotrypsin. Comparison of the k o (catalytic coefficient) of this preparation with that of a similar preparation which had never been exposed to reductant showed a 53% recovery of esterolytic activity towards N-benzoyl-L-tyrosine ethyl ester. Values of K m and K o for non-reduced, matrix-bound zymogen, following its activation to chymotrypsin, were 12.8 × 10 −5 M and 11.0 sec −1, respectively. Corresponding values for reduced, air-reoxidized preparations were 6.8 × 10 −5 M and 3.1 sec −1.

Human pancreatic enzymes: Amino acid sequence of the A-chain of human chymotrypsin II

The A-chain of human chymotrypsin II has been isolated after oxidation of the native protein with performic acid. This peptide contains fourteen residues, one more than the A-chain of bovine δ-chymotrypsin and one less than the A-chain of bovine π-chymotrypsin. The sequence is identical to that found for the first fourteen residues of bovine chymotrypsinogen A with the exception that microheterogeneity was observed at residue six. It would appear that the production of human chymotrypsin II in activated extracts of human pancreas is due, in part, to carboxypeptidase B digestion of a π-chymotrypsin precursor.

Modification of protein properties by change in charge. Succinylated chymotrypsinogen.

Succinylated derivatives of bovine chymotrypsinogen A and α‐chymotrypsin were prepared by treatment of the native proteins with succinic anhydride; 60% of the 6‐amino groups of the 13 lysine residues form stable succinylated products. The large negative charge at neutral pH is reflected in electrophoretic and titration behavior but absorption spectra, rotatory‐dispersion spectra and reversible unfolding behavior indicate that the succinylated species are conformationally similar to the parent proteins. Succinylated chymotrypsin is active as an esterase with N‐acetyl‐l‐tryptophan ethyl ester with a small decrease in the maximum‐velocity parameter. The pH dependence suggests that three ionizing groups influence catalytic behavior. The apparent pKa values analyzed in terms of three groups are shifted relative to α‐chymotrypsin. Thermal‐unfolding studies of succinylated chymotrypsinogen demonstrate the presence of sub‐states near pH 7 but is consistent with two‐state behavior at other pH values.

Preparation and properties of three-chain neochymotrypsinogens

Porcine chymotrypsin C is able to hydrolyze in native bovine chymotrypsinogen A bonds 146 and 148 and also bonds 10 and 13 situated a few residues apart from the “activating” bond Arg 15Ile 16. Hydrolysis of bond 13 does not activate chymotrypsinogen. But it is compatible with subsequent activation of the molecule and, therefore, gives rise to a neochymotrypsinogen with three open chains. Residues Tyr 146 and Arg 145 can be removed from the above neochymotrypsinogen by the combined action of carboxypeptidases A and B, without impairing the activability of the molecule. This observation does not confirm the postulation according to which residue Arg 145 plays a role during chymotrypsinogen activation by trypsin.