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 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 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.

pH-dependence of the fast step of maltose hydrolysis catalysed by glucoamylase G1 from Aspergillus niger

2000 ◽  
Vol 349 (2) ◽  
pp. 623-628 ◽  
Author(s):  
Ulla CHRISTENSEN
Keyword(s):  
Aspergillus Niger ◽  
Ph Dependence ◽  
Acid Group ◽  
Reaction Scheme ◽  
Free Enzyme ◽  
Pka Values ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Range ◽  
State Kinetic

The presteady-state kinetic parameters of the interaction of wild-type glucoamylase from Aspergillus niger (EC 3.2.1.3) with maltose were obtained and analysed in the pH range 3-7 with intervals of 0.25 pH units. In all cases the following three-step reaction scheme was found to apply. E+S ES1 ES2 E+P The general result of the analysis of the presteady-state kinetics is that glucoamylase G1 is affected by the protonation states of three groups, with pKa values of 2.7, 4.5 and 5.7 in the free enzyme and of 2.7, 4.75 and 6.5 in the first enzyme-substrate complex. The protonation of the group in the enzyme-substrate complex with a pKa 6.5 had no effect on k2 (1640 s-1) or k-2 (20±4 s-1), but resulted in a stronger enzyme-substrate interaction, due to a decrease of K1 from 40 to 6.3 mM. In other words, when the substrate is bound, the pKa of the acid group changes to increase the fraction of reactive enzyme. Since this pKa parallels that of the Michaelis complex, known from the pH-dependence of kcat, the group in question is most probably the catalytic acid Glu-179. Protonation of Glu-179 thus is of no importance in the second step, clearly indicating that this step represents a conformational change and not the actual hydrolysis step of the reaction. Protonation of the pKa = 4.75 group leads to a small decrease in k2 to 1090 s-1, and also to minor changes in K1. The group with pKa = 2.7 leads to a major decrease of k2, of which the limit may be zero, but shows no effect on K1. Thus no difference is seen between the pKa values of the free enzyme and of the first enzyme-substrate complex at low pH.

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.

pH Effects in trypsin catalysis

1969 ◽  
Vol 47 (21) ◽  
pp. 4021-4029 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Complex Formation ◽  
Ph Dependence ◽  
Specific Rotation ◽  
Ph Effects ◽  
Ph Values ◽  
A Value ◽  
Available Information ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Covalent Complex

A kinetic study has been made of the trypsin-catalyzed hydrolysis of N-benzoyl-L-alanine methyl ester, at pH values ranging from 6 to 10. The substrate concentrations varied from 1.7 × 10−3 to 4.3 × 10−2 M. From the rates were calculated, at each pH, values of [Formula: see text] (corresponding to [Formula: see text]), [Formula: see text] (corresponding to [Formula: see text]) and [Formula: see text] The specific levorotation of trypsin was measured and found to vary with pH in the pH region 5–11, the change in specific rotation following the ionization of a single group with pK(app) of 9.4. At pH 11 the specific rotation of trypsin, its zymogen, and its phosphorylated derivative were approximately the same, suggesting similar conformations for all three forms of the protein.The kinetic results on the acid side were very similar to those obtained by other investigators for chymotrypsin; they imply that there is a group of [Formula: see text] in the free enzyme, presumably the imidazole function of a histidine residue, and that this group is involved in acylation and deacylation, which can only occur if it is unprotonated. The behavior on the basic side was found to be different from that with chymotrypsin revealing a decrease in [Formula: see text] at high pH corresponding to a value of [Formula: see text] whereas [Formula: see text] showed sigmoid pH-dependence. An interpretation of these results that is consistent with all available information is that a group of [Formula: see text] (presumably the —NH3+ function of the terminal isoleucine) controls the conformation and thereby the activity of the enzyme at different stages of complex formation. In contrast to chymotrypsin, the pK of this ionizing group appears to be generally lowered by covalent complex formation between trypsin and its substrates.

scholarly journals An Improved Procedure for the Preparation of Thrombin Low Molecular Weight Substrates - Peptide p-Nitroanilides

2018 ◽  
Vol 1 (4) ◽  
pp. e00057 ◽  
Author(s):  
A.A Chistov ◽  
A.V. Talanova ◽  
M.V. Melnikova ◽  
S.S. Kuznetsova ◽  
E.F. Kolesanova
Keyword(s):  
Molecular Weight ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Potassium Sulfate ◽  
Polymer Support ◽  
Low Molecular Weight ◽  
Chromogenic Substrate ◽  
Peptide Substrates ◽  
Terminal Amino ◽  
Hydrolysis Of

Low molecular weight chromogenic thrombin peptide substrates, p-nitroanilides of short peptides protected at their N-terminal amino group, were prepared by solid-phase peptide synthesis on polystyrene-divinylbenzene polymer with trityl groups with preliminary attached p-phenylene diamine moiety. After the cleavage from the resin peptide p-aminoanilides were mildly oxidized to p-nitroanilides with the mixture of potassium sulfate and persulfate. Adsorption onto polymer support Bio-Beads SM-2 with further elution by acetonitrile allowed easy separating peptide p-nitroanilides from the oxidizer and obtaining the thrombin chromogenic substrate preparations with the target substance contents of not less than 95% and yields of 30-40%. Thrombin effectively catalyzed hydrolysis of the prepared substrates with KM and Vmax values of 29-134 mM and 0.03-1/16 mM/s, respectively.

scholarly journals The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

1993 ◽  
Vol 290 (1) ◽  
pp. 15-19 ◽  
Author(s):  
A Lewendon ◽  
W V Shaw
Keyword(s):  
Chemical Modification ◽  
Ph Dependence ◽  
Thiol Group ◽  
Chloramphenicol Acetyltransferase ◽  
Histidine Residue ◽  
Pka Values ◽  
Primary Hydroxy Group ◽  
Pka Value ◽  
Catalytic Role ◽  
Kinetics Of

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

scholarly journals A systematic series of synthetic chromophoric substrates for aspartic proteinases

1986 ◽  
Vol 237 (3) ◽  
pp. 899-906 ◽  
Author(s):  
B M Dunn ◽  
M Jimenez ◽  
B F Parten ◽  
M J Valler ◽  
C E Rolph ◽  
...  
Keyword(s):  
Structure Function ◽  
Peptide Bond ◽  
Water Soluble ◽  
Animal Origin ◽  
Aspartic Proteinases ◽  
Peptide Substrates ◽  
Representative Selection ◽  
Micro Organisms ◽  
Hydrolysis Of ◽  
Selection Of

The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.

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