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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha. 3 98

The role of several active site residues of alpha-chymotrypsin in the prototypical refolding transition between active and inactive forms of this enzyme is examined using chemical modification. Oxidation of Met-192 to the sulfoxide results in a derivative which remains entirely in an active state from pH 6 to 9. The derivative becomes inactive only at high pH with pKa = 10.3, delta H0 = 9.5 kcal and delta S0 = -15 eu., indicating the sulfoxide group supplies about 2.1 kcal of active state stabilization relative to the unoxidized methionine side chain. The refolding transition of N-methyl-His-57-alpha-chymotrypsin, in which a nitrogen of the "charge relay" histidine is methylated, displays one ionization process with an apparent pKa of 9.45. The absence of an additional ionization process with a pKa near 7 provides evidence that one of the ionizations in the six state mechanism which describes this transition in alpha-chymotrypsin is linked to the charge relay system. We also demonstrate, using alpha-chymotrypsin, Met-192-sulfoxide-alpha-chymotrypsin and N-methyl-His-57-alpha-chymotrypsin, that the 230 nm circular dichroism band is a quantitative probe of the active-inactive equilibrium, although the chromophore or chromophores responsible for this and another very large negative band at 202 nm have not been identified. Circular dichroism was used to observe the active-inactive equilibrium in methan sulfonyl-alpha-chymotrypsin and phenylmethane sulfonyl-alpha-chymotrypsin. The enhanced stability of the active state of these derivatives relative to alpha-chymotrypsin can be rationalized in terms of steric effects in the substrate side chain binding site.
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PMID:The effects of chemical modification on the refolding transition of alpha-chymotrypsin. 3 46

The effect of gamma irradiation (60Co) of different varieties and breeding lines of dry field beans (Phaseolus vulgaris) on chick growth was determined using a chick growth assay in which the diet contained approximately 50% beans. Total protein (N X 6.25) in beans was not changed appreciably by irradiation (21 Mrad) but protein solubility in water was decreased. Irradiation increased in vitro enzymatic digestibility of bean protein by pepsin and by a mixture of trypsin, chymotrypsin and peptidase. In the bioassay the diet was formulated to derive half of the total protein (22.6%) from beans. Autoclaved Pinto and Pink beans gave significantly better growth than Red Mexican and White Pea beans. The differences between Red Mexican and White Pea beans were not significant except for Red Mexican breeding line number RS-59. The nutritional value of all varieties of beans, based on chick growth, was significantly improved by gamma irradiation. The irradiation treatment of beans tended to increase nitrogen retention by chicks and decrease uric acid nitrogen excretion in relation to nitrogen intake.
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PMID:Effect of gamma irradiation on nutritional value of dry field beans (Phaseolus vulgaris) for chicks. 44 72

Pancreatic secretion of lipase and chymotrypsin in response to elemental diets and a crushed food homogenate was studied in normal subjects. The solutions were infused at constant flow rates at the ligament of Treitz with polyethylene glycol as a nonabsorbable marker. A triple lumen tube was used, enabling collection of secretions at 35 and 70 cm from the infusion point. The results show that a crushed food homogenate has a greater stimulative effect on pancreatic enzyme secretion than the elemental solutions and that this can be directly related to its greater nitrogen content. The osmolality of the infused solutions does not appear to be important. The relative merits of the solutions tested and total parental nutrition in reducing pancreatic enzyme secretion are discussed.
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PMID:Effect of continuous jejunal perfusion of elemental and complex nutritional solutions on pancreatic enzyme secretion in human subjects. 63 40

Lysosomal fraction was isolated from rat liver by density gradient centrifugation after pervious loading of lysosomes in vivo with Triton WR-1339. Tritosome preparations were incubated at 37 degrees C and pH 5 for 24 hr with purified human ceruloplasmin or haptoglobin. After this period approximately 20% of total alpha amino nitrogen was released from ceruloplasmin and over 40% from haptoglobin. This was accompanied by loss of peroxidase activity of haptoglobin (in complex with haemoglobin), while enzymatic activity of ceruloplasmin remained unaltered. Removal of sialic acid by neuraminidase had no effect on digestion of ceruloplasmin by rat liver tritosomes. Both glycoproteins were resistant to horse leucocyte proteinases and pancreatic eleastase but were easily inactivated by trypsin and chymotrypsin.
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PMID:Degradation and inactivation of ceruloplasmin and haptoglobin by rat liver lysosomes and some other proteinases. 65 34

The influence of the leaving group on the reactivity of specific anilides in alpha-chymotrypsin-catalyzed hydrolysis (chymotrypsin, EC 3.4.21.2) involves both its binding to the enzyme (steric effect) and electronic nature (electronic effect). These effects are considered in terms of the stereoelectronic theory for the formation and cleavage of the tetrahedral intermediate in acyltransfer reactions. The application of this theory to the enzyme hydrolysis leads to the conclusion that the nature of the reaction products and the effectiveness of the catalysis are controlled by the orientation of the leaving group nitrogen lone pair orbital. The leaving group binding affects the formation of a reactive conformation of the enzyme tetrahedral intermediate that is presumed to intervene between the Michaelis complex and the acylenzyme. The steric and electronic effects could be separated in a straightforward fashion only in the case of equal binding of the leaving groups to the leaving-group-binding site of alpha-chymotrypsin.
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PMID:Catalysis and leaving group binding in anilide hydrolysis by chymotrypsin. 71 54

Pyrolysis gas chromatography (PGC) has been shown to be useful for differentiating enzymes. The enzymes alpha-chymotrypsin, lactate dehydrogenase, catalase, and urease were easily "fingerprinted" on a 1.8 m 0.5% Carbowax 20 M column. Also, in some cases, isoenzymes of lactate dehydrogenase could be distinguished. Based on the pyrolyses of the free aromatic amino acids, four major enzyme pyrolysis peaks were tentatively identified as organic compounds derived from tyrosine and tryptophan. The use of a nitrogen-selective detector in conjunction with the FID and measurement of peak retention times by computer on three different types of columns permitted confirmations of peak identity.
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PMID:Pyrolysis gas chromatography of enzymes. 73 Aug 12

A series of N-acetylated tetra- to heptapeptide amides has synthesized for the study of enzyme-substrate interactions beyond the S1' subsite in Streptomyces griseus Protease 3 (SGP3) and alpha-chymotrypsin (EC 3.4.21.1). Evidence was obtained that S2'-P2' enzyme-substrate interactions can play a significant role for the rate of substrate hydrolysis in both enzymes. No important interaction could be demonstrated beyond the nitrogen atom of residue P3'. This provides supplementary evidence that the active site of SGP3 extends over 6-7 subsites and that of alpha-chymotrypsin over 5-6 subsites. SGP3 is a considerably more efficient protease than alpha-chymotrypsin, kcat/Km being approximately 5-10(6) S-1-M-1 for the best substrates, thus being about 100 times higher than for alpha-chymotrypsin. However, an analysis of the kinetic data indicates that, for both enzymes, the acylation rates for the best peptide substrates approach their deacylation rates.
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PMID:The active centers of Streptomyces griseus protease 3 and alpha-chymotrypsin. Enzyme-substrate interactions beyond subsite S'1. 82 30

The time-courses of proteolytic activities in pancreatic tissue and the contents of the small intestine (the intestinal contents) were determined in rats maintained on a diet containing 30% of various proteins after a switchover from a diet containing 12% casein. 1. The proteolytic activity of the pancreatic tissue quickly responded to change of dietary proteins--within 1 to 6 days--with respect to organ weight, nitrogen content and proteolytic activity, in rats receiving diets containing 30% casein, ovalbumin, lactalbumin, gluten, gelatin or zein. 2. However, the proteolytic activity in the intestinal contents did not necessarily coincide with the pancreatic digestive function; an approximately threefold increase of enzyme activity was demonstrated on the fifth day of feeding in rats receiving gluten. 3. The proteolytic activity in the intestinal contents returned to the initial level on the eighth day in the gluten-fed rats, but those rats maintained on a lysine-supplemented gluten diet exhibited no such elevation of proteolytic activity. 4. No significant difference in pancreatic composition was shown up to the eighth day between the group receiving gluten alone in diet and that receiving the same diet but supplemented with lysine, under the condition of equally restricted food intake. Intestinal trypsin and chymotrypsin levels, however, were higher in the gluten-fed rats, suggesting that the depressed rate of enzyme inactivation in the small intestine might be the principal cause of the finding described under (2) above.
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PMID:Effect of dietary protein on proteolytic activities in the pancreatic tissue and contents of the small intestine in rats. 121 81

alpha-Chymotrypsin serves as a sole carbon source, sole nitrogen source, and as sole carbon plus nitrogen source for wild-type Escherichia coli in a totally defined medium. Hence, a mammalian host for E. coli may supply the necessary carbon and nitrogen nutrients for the microorganism. Growth is most rapid when chymotrypsin is a sole nitrogen source and least rapid with chymotrypsin as a carbon source. The approximate doubling times for E. coli utilizing chymotrypsin as a nitrogen source, carbon plus nitrogen source, and carbon source are 1.6, 4.6, and 11.3 h, respectively. The activity of the residual enzyme in the culture supernates falls off asymptotically over the source of time, as followed by cleavage of glutaryl-L-phenylalanine-p-nitroanilide. Chymotrypsin hydrolyzes succinyl-L-ala-L-ala-p-nitroanilide, the elastase substrate, to some extent. Peptidases do not appear to be secreted that hydrolyze such model substrates as benzoyl-DL-arginine-p-nitroanilide, the tryptic and cathepsin B substrate, L-leucine-p-nitroanilide, the leucine amino-peptidase substrate, or L-lysine-p-nitroanilide, the aminopeptidase B substrate. Growth of E. coli is generally directly related to the loss of chymotryptic activity in the medium. Hence, autolysis of chymotrypsin, i.e., self-degradation, is an important factor for the availability of degradation products of the enzyme to the bacterium for growth purposes. Accordingly, the degradation of a host protein by autolysis presents an opportunity for E. coli to survive during periods of host nutritional crisis by utilization of the degradation peptides that are produced during autolysis.
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PMID:Utilization of chymotrypsin as a sole carbon and (or) nitrogen source by Escherichia coli. 161 54


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