scholarly journals The catalytic activity of pig pepsin C towards small synthetic substrates

1979 ◽  
Vol 179 (1) ◽  
pp. 239-246 ◽  
Author(s):  
C A Auffret ◽  
A P Ryle
Keyword(s):  
Catalytic Activity ◽  
General Formula ◽  
Competitive Inhibition ◽  
Ph Dependence ◽  
Small Peptides ◽  
Pka Values ◽  
A Minor ◽  
Hydrolysis Of

A series of small peptides has been synthesized and used to investigate the activity of a minor pig pepsin, pepsin C (EC 3.4.23.3). The peptides had the general formula A-Leu-Val-His-B. B was either OMe, NH2 or OH. With B = NH2 hydrolysis (kcat./Km) at 37 degrees C and pH 2.07 increased as A was Ac-Ala, Ac-Tyr, Ac-Phe and Ac-Ala-Phe. The pH dependence of the hydrolysis of Ac-Phe-Leu-Val-His-NH2 indicated the apparent pKa values of two catalytically important groups on the enzyme as 1.42 and 4.88. Inhibition of the hydrolysis of the same peptide by Ac-Phe at pH 3.01 showed a form of mixed non-competitive inhibition. Hydrolysis of Ac-Tyr-Leu-Val-His-OMe and the corresponding amide showed non-classical kinetics, which are discussed in terms of a substrate-activating mechanism. The results are discussed with reference to observations made by other workers on pig pepsin A.

scholarly journals Chemical synthesis and papain-catalysed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide

1985 ◽  
Vol 226 (2) ◽  
pp. 601-606 ◽  
Author(s):  
N E Mackenzie ◽  
J P G Malthouse ◽  
A I Scott
Keyword(s):  
Chemical Synthesis ◽  
Ph Dependence ◽  
Low Ph ◽  
Pka Values ◽  
Tetrahedral Intermediate ◽  
Ph Values ◽  
Km Value ◽  
Hydrolysis Of

The chemical synthesis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide (Z-Lys-pNA) is described in detail. The pH-dependence of the catalytic parameters kcat,' Km and kcat./Km for the papain-catalysed hydrolysis of Z-Lys-pNA are determined. kcat. and Km are pH-independent between pH 5 and pH 7.42, but the pH-dependence of kcat./Km is bell-shaped, decreasing at high and low pH values with pKa values of 7.97 and 4.40 respectively. The catalytic parameters and their pH-dependence are shown to be similar to those reported for other anilide substrates and it is concluded that the Km value of 0.01 mM previously reported [Angelides & Fink (1979) Biochemistry 18, 2355-2369] is incorrect. The possibility of accumulating a tetrahedral intermediate during the papain-catalysed hydrolysis of Z-Lys-pNA is discussed.

scholarly journals The pH-dependence of pepsin-catalysed reactions

1969 ◽  
Vol 113 (2) ◽  
pp. 353-362 ◽  
Author(s):  
A. J. Cornish-Bowden ◽  
J. R. Knowles
Keyword(s):  
Continuous Monitoring ◽  
Ph Dependence ◽  
Preceding Paper ◽  
Free Enzyme ◽  
Peptide Substrates ◽  
Pka Values ◽  
Hydrolysis Of

1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pKa values of 1·1 and 3·5 in the plots of k0/Km versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pKa values of 1·0 and 4·7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pKa values of 1·0 and 4·7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pKa values of 3·5 for the dependence of k0/Km for these materials.

scholarly journals The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre

1999 ◽  
Vol 338 (1) ◽  
pp. 235-239 ◽  
Author(s):  
Manuel MORILLAS ◽  
Martin L. GOBLE ◽  
Richard VIRDEN
Keyword(s):  
Rate Constant ◽  
Conformational Transition ◽  
Ph Dependence ◽  
Penicillin Acylase ◽  
Penicillin G ◽  
Pka Values ◽  
Energy Compensation ◽  
Guanidinium Group ◽  
Kinetics Of ◽  
Hydrolysis Of

Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a kcat of 0.8 s-1 and a Km of 10 µM at pH 7.5 and 20 °C. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s-1 and a subsequent deacylation step with a rate constant of 0.81 s-1. Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to KS was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At 20 °C, the pH-dependence of kcat was similar to that of kcat/Km, indicating that formation of the acyl-enzyme did not affect the pKa values (6.5 and 9.0) of an acidic and basic group in the active enzyme. The heats of ionization deduced from values of pKa for kcat, which measures the rate of deacylation, are consistent with α-amino and guanidinium groups whose pKa values are decreased in a non-polar environment. It is proposed that, for catalytic activity, the α-amino group of the catalytic SerB1 and the guanidinium group of ArgB263 are required in neutral and protonated states respectively.

Immobilized Nanoparticles-Mediated Enzymatic Hydrolysis of Cellulose for Clean Sugar Production: A Novel Approach

2019 ◽  
Vol 15 (3) ◽  
pp. 296-303 ◽  
Author(s):  
Swapnil Gaikwad ◽  
Avinash P. Ingle ◽  
Silvio Silverio da Silva ◽  
Mahendra Rai
Keyword(s):  
Catalytic Activity ◽  
Enzymatic Hydrolysis ◽  
Immobilized Enzyme ◽  
Free Enzyme ◽  
Magnetic Nanomaterials ◽  
Sugar Production ◽  
Cellulase Enzyme ◽  
Enzymatic Hydrolysis Of Cellulose ◽  
Hydrolysis Of Cellulose ◽  
Hydrolysis Of

Background: Enzymatic hydrolysis of cellulose is an expensive approach due to the high cost of an enzyme involved in the process. The goal of the current study was to apply magnetic nanomaterials as a support for immobilization of enzyme, which helps in the repeated use of immobilized enzyme for hydrolysis to make the process cost-effective. In addition, it will also provide stability to enzyme and increase its catalytic activity. Objective: The main aim of the present study is to immobilize cellulase enzyme on Magnetic Nanoparticles (MNPs) in order to enable the enzyme to be re-used for clean sugar production from cellulose. Methods: MNPs were synthesized using chemical precipitation methods and characterized by different techniques. Further, cellulase enzyme was immobilized on MNPs and efficacy of free and immobilized cellulase for hydrolysis of cellulose was evaluated. Results: Enzymatic hydrolysis of cellulose by immobilized enzyme showed enhanced catalytic activity after 48 hours compared to free enzyme. In first cycle of hydrolysis, immobilized enzyme hydrolyzed the cellulose and produced 19.5 ± 0.15 gm/L of glucose after 48 hours. On the contrary, free enzyme produced only 13.7 ± 0.25 gm/L of glucose in 48 hours. Immobilized enzyme maintained its stability and produced 6.15 ± 0.15 and 3.03 ± 0.25 gm/L of glucose in second and third cycle, respectively after 48 hours. Conclusion: This study will be very useful for sugar production because of enzyme binding efficiency and admirable reusability of immobilized enzyme, which leads to the significant increase in production of sugar from cellulosic materials.

scholarly journals Encapsulated NOLA™ Fit 5500 Lactase—An Economically Beneficial Way to Obtain Lactose-Free Milk at Low Temperature

Catalysts ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 527
Author(s):  
Katarzyna Czyzewska ◽  
Anna Trusek
Keyword(s):  
Low Temperature ◽  
Storage Temperature ◽  
Competitive Inhibition ◽  
Caloric Content ◽  
Substrate Diffusion ◽  
Galactose Inhibition ◽  
The Subject ◽  
And Storage ◽  
Food Processes ◽  
Hydrolysis Of

The current requirements of industrial biocatalysis are related to economically beneficial and environmentally friendly processes. Such a strategy engages low-temperature reactions. The presented approach is essential, especially in food processes, where temperature affects the quality and nutritional value foodstuffs. The subject of the study is the hydrolysis of lactose with the commercial lactase NOLA™ Fit 5500 (NOLA). The complete decomposition of lactose into two monosaccharides gives a sweeter product, recommended for lactose intolerant people and those controlling a product’s caloric content. The hydrolysis reaction was performed at 15 °C, which is related to milk transportation and storage temperature. The enzyme showed activity over the entire range of substrate concentrations (up to 55 g/L lactose). For reusability and easy isolation, the enzyme was encapsulated in a sodium alginate network. Its stability allows carrying out six cycles of the complete hydrolysis of lactose to monosaccharides, lasting from two to four hours. During the study, the kinetic description of native and encapsulated NOLA was conducted. As a result, the model of competitive galactose inhibition and glucose mixed influence (competitive inhibition and activation) was proposed. The capsule size does not influence the reaction rate; thus, the substrate diffusion into capsules can be omitted from the process description. The prepared 4 mm capsules are easy to separate between cycles, e.g., using sieves.

The pH Dependence of the Competitive Inhibition of Fumarase1,2

1960 ◽  
Vol 82 (20) ◽  
pp. 5482-5488 ◽  
Author(s):  
Paul W. Wigler ◽  
Robert A. Alberty
Keyword(s):  
Competitive Inhibition ◽  
Ph Dependence

Enzymatic deglycosylation of porcine thyroid peroxidase: effects on catalytic activity and immunoreactivity

1991 ◽  
Vol 124 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Egberto G. Moura ◽  
Carmen C. Pazos-Moura ◽  
Naokata Yokoyama ◽  
Martha L. Dorris ◽  
Alvin Taurog
Keyword(s):  
Catalytic Activity ◽  
Molecular Mass ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Minor Role ◽  
Relative Molecular Mass ◽  
Thyroid Peroxidase ◽  
Autoimmune Thyroid ◽  
Tryptic Fragment ◽  
A Minor

Abstract Thyroid peroxidase is a heme-containing, membrane-bound, glycoprotein enzyme that catalyzes iodination and coupling in the thyroid gland. It is also the antigen for microsomal autoantibodies that are commonly found in the serum of patients with autoimmune thyroid disease. We examined the effect of deglycosylation on the catalytic functions and the immunoreactivity of this enzyme. A highly purified, solubilized, large tryptic fragment of porcine thyroid peroxidase, retaining all of the N-linked glycosylation sites of the native enzyme and displaying full catalytic activity was used. It was deglycosylated by treatment with N-glycanase under nondenaturing conditions. The loss in relative molecular mass after treatment, determined by gel electrophoresis, was about 75% of the estimated molecular weight of the glycan portion of porcine thyroid peroxidase. Lectin blots performed with horseradish peroxidase-conjugated concanavalin A showed a similar loss in relative molecular mass but some residual carbohydrate. The intensity of the carbohydrate stain was consistent with the loss of about 75% of the glycans. Despite this loss, three different assays for catalytic activity of porcine thyroid peroxidase were not significantly decreased. Immunoreactivity measured by immunoblotting and by enzyme-linked immunosorbent assay was also unimpaired. These findings suggest that N-glycanase-sensitive glycans in porcine thyroid peroxidase do not act as antigenic determinants and play a minor role, if any, in catalytic activity and, presumably therefore, in the maintenance of protein conformation.

Functional specificity of jejunal brush-border pteroylpolyglutamate hydrolase in pig

1991 ◽  
Vol 260 (6) ◽  
pp. G865-G872 ◽  
Author(s):  
C. J. Chandler ◽  
D. A. Harrison ◽  
C. A. Buffington ◽  
N. A. Santiago ◽  
C. H. Halsted
Keyword(s):  
Brush Border ◽  
Protein Band ◽  
Major Protein ◽  
Functional Specificity ◽  
Major Protein Band ◽  
Regional Location ◽  
Minor Protein ◽  
A Minor ◽  
Hydrolysis Of

To determine the functional specificity of intestinal brush-border pteroylpolyglutamate hydrolase (PPH), we compared the regional location of in vivo hydrolysis of pteroyltriglutamate (PteGlu3) with the location of activity and immunoreactivity of the enzyme in the pig. After in vivo incubations, PteGlu3 hydrolytic products were recovered from intestinal segments in the jejunum but not from the ileum. Brush-border PPH activity in fractionated mucosa was 10-fold greater in the jejunum than in the ileum, whereas the activity of intracellular PPH was increased in the distal ileum. Antibodies to purified brush-border PPH identified a major protein band at 120 kDa and a minor protein band at 195 kDa in solubilized jejunal brush border. Immunohistochemistry identified the enzyme only on the brush-border surface of the jejunum, whereas an immunoblot of solubilized brush-border membranes identified brush-border PPH in the jejunum but not in the ileum. The parallel of the regional location of in vivo hydrolysis of PteGlu3 with the location of brush-border PPH activity and immunoreactivity demonstrates the functional specificity of this enzyme in folate digestion.

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