Mechanisms of action of trypsin and chymotrypsin

Kasserra, H. P.; Laidler, Keith J.

doi:10.1139/v69-669

Cited by 26 publications

(8 citation statements)

References 11 publications

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“…The pH dependence of k cat / K M reflects ionizations in either the free enzyme or the free substrate. Previous studies with other substrates have detected similar free enzyme p K a values of ∼7 and ∼4. ,− …”

Section: Resultsmentioning

confidence: 59%

“…The pH dependence of k cat / K M showed that the active group was A 2– in eq and that it was inctivated by protonation to form AH – and AH 2 (eq ) The free enzyme p K a values 6.75 ± 0.09 and p K b = 4.10 ± 0.13 obtained from the pH dependence of k cat / K M and 1/ K s (Table ) have been attributed to the trypsin residues histidine-57 and aspartate-189, respectively. ,− In both cases, it is the ionized form of these groups that are catalytically active (structure a1 in Scheme ). Histidine-57 is part of the catalytic triad and its ionized form acts as a general base catalyst during catalysis (b1 in Scheme ).…”

Section: Discussionmentioning

confidence: 99%

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Kinetic Studies of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of N-α-benzyloxycarbonyl-l-lysine-p-nitroanilide: Mechanism of Trypsin Catalysis

Malthouse

2020

ACS Omega

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The pH dependence of the trypsin-catalyzed hydrolysis of N-α-benzyloxycarbonyl-L-lysine p-nitroanilide has been studied at 25 °C. k cat /K M was maximal at alkaline pH values but decreased with decreasing pH. k cat /K M was dependent on free enzyme pK a values of 6.75 ± 0.09 and 4.10 ± 0.13, which were assigned to the ionization of the active site histidine-57 and aspartate-189, respectively. Protonation of either group abolished catalytic activity. k cat is shown to equal the acylation rate constant k 2 over the pH range studied. k 2 decreased on the protonation of two groups with pK a values of 4.81 ± 0.15 and 4.23 ± 0.19. We assign the pK a of 4.23 to the ionization of the aspartate-189 residue and the pK a of 4.81 to the oxyanion of the tetrahedral intermediate formed during acylation. We conclude that during acylation, breakdown of the catalytic tetrahedral intermediate is rate-limiting and that there is a strong interaction between the imidazolium ion of histidine-57 and the oxyanion of the catalytic tetrahedral intermediate, which perturbs their pK a values. From the pH dependence of k 3 , we conclude that deacylation depends on a pK a of 6.41 ± 0.22 and that the ionization of the carboxylate group of aspartate-189 does not have a significant effect on the rate of deacylation (k 3 ). A catalytic mechanism is proposed to explain the pH dependence of catalysis.

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Section: Resultsmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Kinetic Studies of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of N-α-benzyloxycarbonyl-l-lysine-p-nitroanilide: Mechanism of Trypsin Catalysis

Malthouse

2020

ACS Omega

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“…Herein, proteins are digested into peptides by use of a protease, usually trypsin, which predominantly cleaves the protein at the carboxyl side of lysine and arginine (except when followed by proline) [23]. These peptides are characteristic, specific and unique for a given protein: identifying them means identifying the protein as a whole and as such, the protein binder source it constitutes.…”

Section: Introductionmentioning

confidence: 99%

Tryptic peptide analysis of protein binders in works of art by liquid chromatography–tandem mass spectrometry

Fremout

Dhaenens

Saverwyns

et al. 2010

Analytica Chimica Acta

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Comprehension of the materials, such as binding media, used by artists is of uttermost importance in restoration and in art historical studies. The most frequently used binders are drying oils and proteins; in this study focus is placed on proteins. Most actual methods for protein binder identification are based on complete hydrolyzation of the protein matter into its amino acids and separation/detection with gas chromatographymass spectrometry (GC-MS) or high performance liquid chromatography (HPLC) after derivatization. Because amino acids itself are not characteristic for a protein, identification is often based on the relative amount of 7 stable amino acids. In the current study a proteomics approach was used, in which the proteins were digested enzymatically into peptides using trypsin before being separated and detected by liquid chromatography -electronspray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Mascot (Matrix Science) was used to analyze the resulting data and for protein identification. This way, amino acid sequences could be studied that retain much more information about the proteins, their degradation and pigment-binder interactions. The protein content of homemade paint samples was extracted using different methods and analysed to select the best extraction strategy based on the number of peptides that were identified. A large dataset of 4 binders (animal glue, egg yolk, egg white and casein), mixed with 10 common pigments with different chemical properties was used to study the influence of pigments on the extraction method.Analytical characteristics of the selected method were determined. Finally the method was applied to historic paint samples. The results were compared with those obtained by traditional amino acid analysis methods.

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“…Chymotrypsin is one of the most well studied peptidases (enzymes that catalyze the cleavage of peptide bonds) [130][131][132]. Instead of using water as nucleophile, the alcohol side chain of serine-195 is used (Scheme 16).…”

Section: Enzyme-catalyzed Acyl Transfersmentioning

confidence: 99%

Organocatalysis: Fundamentals and Comparisons to Metal and Enzyme Catalysis

et al. 2016

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Catalysis fulfills the promise that high-yielding chemical transformations will require little energy and produce no toxic waste. This message is carried by the study of the evolution of molecular catalysis of some of the most important reactions in organic chemistry. After reviewing the conceptual underpinnings of catalysis, we discuss the applications of different catalysts according to the mechanism of the reactions that they catalyze, including acyl group transfers, nucleophilic additions and substitutions, and C-C bond forming reactions that employ umpolung by nucleophilic additions to C=O and C=C double bonds. We highlight the utility of a broad range of organocatalysts other than compounds based on proline, the cinchona alkaloids and binaphthyls, which have been abundantly reviewed elsewhere. The focus is on organocatalysts, although a few examples employing metal complexes and enzymes are also included due to their significance. Classical Brønsted acids have evolved into electrophilic hands, the fingers of which are hydrogen donors (like enzymes) or other electrophilic moieties. Classical Lewis base catalysts have evolved into tridimensional, chiral nucleophiles that are N-(e.g., tertiary amines), P-(e.g., tertiary phosphines) and C-nucleophiles (e.g., N-heterocyclic carbenes). Many efficient organocatalysts bear electrophilic and nucleophilic moieties that interact simultaneously or not with both the electrophilic and nucleophilic reactants. A detailed understanding of the reaction mechanisms permits the design of better catalysts. Their construction represents a molecular science in itself, suggesting that sooner or later chemists will not only imitate Nature but be able to catalyze a much wider range of reactions with high chemo-, regio-, stereo-and enantioselectivity. Man-made organocatalysts are much smaller, cheaper and more stable than enzymes.

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Mechanisms of action of trypsin and chymotrypsin

Cited by 26 publications

References 11 publications

Kinetic Studies of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of N-α-benzyloxycarbonyl-l-lysine-p-nitroanilide: Mechanism of Trypsin Catalysis

Kinetic Studies of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of N-α-benzyloxycarbonyl-l-lysine-p-nitroanilide: Mechanism of Trypsin Catalysis

Tryptic peptide analysis of protein binders in works of art by liquid chromatography–tandem mass spectrometry

Organocatalysis: Fundamentals and Comparisons to Metal and Enzyme Catalysis

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