scholarly journals Cryoenzymology of trypsin. A detailed kinetic study of the trypsin-catalysed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitrophenyl ester at low temperatures

1983 ◽  
Vol 215 (3) ◽  
pp. 555-563 ◽  
Author(s):  
J P G Malthouse ◽  
A I Scott
Keyword(s):  
Aqueous Media ◽  
Side Chain ◽  
Ambient Temperatures ◽  
Ph Values ◽  
Mechanism Of Catalysis ◽  
The Individual ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester ◽  
Positively Charged

A detailed study of the kinetics of the trypsin (EC 3.4.21.4)-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester in cryosolvents at 0 degrees C and below was undertaken. The pH-dependences of kcat, Km, k+2, k+3 and Ks were determined under cryoenzymological conditions and are compared with previous results [Antonini & Ascenzi (1981) J. Biol. Chem. 256, 12449-12455] obtained in fully aqueous media at ambient temperatures. Below pH 5.0 the kinetics, and presumably the mechanism of catalysis, are not significantly perturbed under cryoenzymological conditions. However, it is shown that below pH 5.0 both Km and Ks are decreased under these conditions but that both are increased at pH 6.7 relative to the results obtained in fully aqueous media at ambient temperatures. The effects of the cryoenzymological conditions on the individual catalytic parameters are discussed. The acylation rate constant, k+2, is essentially constant at pH 4.2 and 5.0 but decreases at lower pH values with an apparent pKa of approx. 4.0. In view of the low enthalpy of ionization associated with this pKa it is suggested that this group is the carboxy group of aspartic acid-189, which binds the positively charged lysine side chain of the substrate. The mechanistic implications of the results for the acylation step are discussed. It is also shown that only at low pH values can significant amounts of acylated trypsin be accumulated.

Long chain ß-aminoethanols as micellar catalysts for the hydrolysis of diphenyl-p-nitrophenyl phosphate. The observation of an unusual enhancement of catalysis in the presence of added polyols

1999 ◽  
Vol 77 (10) ◽  
pp. 1577-1588
Author(s):  
H Oumar-Mahamat ◽  
H Slebocka-Tilk ◽  
R S Brown
Keyword(s):  
Ethylene Glycol ◽  
Aqueous Media ◽  
Polyethylene Glycol 400 ◽  
Ph Values ◽  
Peg 400 ◽  
Nitrophenyl Phosphate ◽  
Aqueous Ethylene Glycol ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Long Chain

The hydrolysis of p-nitrophenyldiphenyl phosphate (PNPDPP, 7) catalyzed by N-dodecylaminoethanol (6a), N-tetradecylaminoethanol (6b), N-methyl-N-tetradecylaminoethanol (6c), and N-hexadecylaminoethanol (6d) was investigated at 25°C at various pH values in aqueous media containing additives. Added ethanol or dimethoxyethane produces very little activity of 6a, b, c toward 7. The highest activities of 6a, b were found at pH 8.0 and 8.5 in solvents containing 20% glycerol or ethylene glycol. Under no conditions was N-hexadecylaminoethanol (6d) found to be active. The kinetics of the reaction of 6b with 7 were also investigated in a medium containing various amounts of glycerol, polyethylene glycol 400 (PEG 400), and water at pH 7.6-8.5. The best catalytic system comprised 3 mM 6b in 20% aqueous ethylene glycol, pH 8.5, which accelerated the hydrolysis of 7 by 1700-fold over the blank rate. Kinetic experiments conducted using [7]/[6b] = 1 and 2 demonstrate that 6b exhibits true turnover catalysis. A mechanism for the catalyzed reaction is proposed.Key words: ß-aminoethanols, hydrolysis, p-nitrophenyldiphenyl phosphate, micelles, polyhydroxy alcohols.

scholarly journals Structure of a Talaromyces pinophilus GH62 arabinofuranosidase in complex with AraDNJ at 1.25 Å resolution

2018 ◽  
Vol 74 (8) ◽  
pp. 490-495 ◽  
Author(s):  
Olga V. Moroz ◽  
Lukasz F. Sobala ◽  
Elena Blagova ◽  
Travis Coyle ◽  
Wei Peng ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulosic Biomass ◽  
Structural Basis ◽  
Side Chain ◽  
Polymeric Substrates ◽  
Hydrolysis Of ◽  
Insight Into ◽  
Positively Charged ◽  
Talaromyces Pinophilus ◽  
Resolution Structure

The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric β-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25 Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a K d of 24 ± 0.4 µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.

Kinetics of acid-assisted hydrolysis of pentachlorophenol in aqueous media

2008 ◽  
Vol 49 (3) ◽  
pp. 361-365 ◽  
Author(s):  
S. M. Kulikov ◽  
B. J. R. Holder
Keyword(s):  
Aqueous Media ◽  
Kinetics Of ◽  
Hydrolysis Of

scholarly journals The effect of temperature on the individual stages of the hydrolysis of non-specific-p-nitrophenol esters by α-chymotrypsin

1977 ◽  
Vol 161 (1) ◽  
pp. 83-92 ◽  
Author(s):  
P A Adams ◽  
E R Swart
Keyword(s):  
Kinetic Studies ◽  
Detailed Comparison ◽  
Effect Of Temperature ◽  
Thermal Transitions ◽  
Arrhenius Plots ◽  
Ph Values ◽  
The Individual ◽  
Hydrolysis Of

Precise studies were performed on the effect of temperature on the rate and equilibrium parameters characterizing the individual stages of the alpha-chymotrypsin-catalysed hydrolysis of non-specific p-nitrophenol esters at pH 7.40 and 8.50. At both pH values the results indicate that a sharp kinetic anomaly is observed in Arrhenius plots of these parameters for the binding and acylation stages of the process, but not for the deacylation stage. Detailed comparison with other kinetic studies was made, and a comparison with thermal transitions observed in alpha-chymotrypsin by using physical techniques was attempted. A detailed discussion of possible causes of the anomalies is given.

scholarly journals Cryoenzymology of trypsin. 13C-n.m.r. detection of an acyl-trypsin intermediate in the trypsin-catalysed hydrolysis of a highly specific substrate at subzero temperature

1984 ◽  
Vol 219 (2) ◽  
pp. 437-444 ◽  
Author(s):  
N E Mackenzie ◽  
J P Malthouse ◽  
A I Scott
Keyword(s):  
Subzero Temperature ◽  
Specific Substrate ◽  
Visible Absorption ◽  
Enzyme Substrate ◽  
First Time ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

The kinetics of the trypsin-catalysed hydrolysis of the highly specific substrate N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester were studied under cryoenzymological conditions by 13C-n.m.r. spectroscopy at pH approx. 3.0. The kinetics of this reaction are shown to be in agreement with similar studies made with the use of u.v.-visible-absorption-spectrophotometric techniques. A combination of 13C-n.m.r. spectroscopy and cryoenzymology has for the first time detected an acyl-trypsin intermediate in the hydrolysis of this highly specific substrate. The advantages and difficulties of using 13C-n.m.r. spectroscopy coupled with cryoenzymology in the detection and characterization of enzyme-substrate intermediates are discussed.

scholarly journals Rapid burst kinetics in the hydrolysis of 4-nitrophenyl acetate by penicillin G acylase from Kluyvera citrophila. Effects of mutation F360V on rate constants for acylation and de-acylation

1996 ◽  
Vol 316 (2) ◽  
pp. 409-412 ◽  
Author(s):  
A Roa ◽  
M L Goble ◽  
J L García ◽  
C Acebal ◽  
R Virden
Keyword(s):  
Rate Constants ◽  
Acetyl Derivative ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Side Chain ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Burst Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1·s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1·s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

Kinetics of a monomolecular reaction between n components

1980 ◽  
Vol 45 (4) ◽  
pp. 1197-1220 ◽  
Author(s):  
Jaromír Jakeš
Keyword(s):  
Reaction Kinetics ◽  
Difficult Case ◽  
System A ◽  
Monomolecular Reaction ◽  
Consecutive Reactions ◽  
Multiple Eigenvalues ◽  
The Individual ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Derivatives Of

The reaction kinetics has been investigated of a general monomolecular reaction between n components, where reactions between some components are reversible and between others irreversible. The reacting components may be divided into groups so that all the components inside one group may change reversibly into each other, while reactions between components of different groups are irreversible. The reaction kinetics for each reversible group may be found similarly to the case where all the reactions are reversible; solutions for the individual reversible groups may be used to obtain solution for the whole system. A solution was also found to a difficult case in which matrices have multiple eigenvalues for irreversible consecutive reactions, namely, for a general case of degeneracy. Formulas are given for the calculation of derivatives of concentrations of the individual components with respect to parameters. The equations thus derived were applied to the reaction kinetics of a polymeranalogous reaction (e.g., hydrolysis of polyacrylonitrile).

Polygalacturonases of Aspergillus flavipes FP-500: A Kinetic Analysis of Batch Culture Systems Based on Unstructured Models

2012 ◽  
Vol 10 (1) ◽  
Author(s):  
Maria Aurora Martinez Trujillo ◽  
Juan S. Aranda Barradas ◽  
Guillermo Aguilar Osorio
Keyword(s):  
Galacturonic Acid ◽  
Culture Media ◽  
Culture Conditions ◽  
Batch Cultures ◽  
Ph Values ◽  
Aspergillus Flavipes ◽  
Production Kinetics ◽  
Pectinolytic Enzymes ◽  
Kinetics Of ◽  
Hydrolysis Of

Abstract Pectin is a widespread complex heteropolysaccharide contained in plants cell wall. The hydrolysis of this natural-occurring polymer is an important process both in food industry and pectin-rich wastewater treatment. Although pectin-degrading enzymes have been produced classically by microbial mold strains, production kinetics of the involved endo- and exo- pectinolytic enzymes is still a challenge in industrial microbiology. In order to assess the pectinases production kinetics, the strain Aspergillus flavipes FP-500 was grown in batch cultures using pectin, glucose or galacturonic acid as limiting substrates. Unstructured models were useful for describing the experimental behavior, and for estimating the kinetic parameters associated to the Logistic, Monod and Luedeking-Piret models. Our results pointed out that the exopolygalacturonases production is basically non-growth associated, suggesting an inducible nature for some exo- isoenzymes and endopolygalacturonases, even if some constitutive activity is postulated. Besides, to identify the combined effect of carbon source and pH on polygalacturonases production, several experiments were developed at different pH culture conditions. Exopectinases produced on glucose were inhibited by culture media acidification, while on galacturonic acid these enzymes are produced mainly at pH values of 5.0 or higher. Exopectinases production on pectin was not importantly affected by the established pH values during the culture. Endopectinases were produced basically at acidic conditions on pectin, but growth on galacturonic acid showed a strong inducing effect on endopectinases at pH 5.0

The kinetics of the reaction of N,N-dimethyl-2-phenylaziridinium ion with bovine erythrocyte acetylcholinesterase

1970 ◽  
Vol 48 (3) ◽  
pp. 244-250 ◽  
Author(s):  
Jocelyn E. Purdie ◽  
R. M. Heggie
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Bovine Erythrocyte ◽  
Acidic Group ◽  
Ph Values ◽  
Order Rate ◽  
Decreasing Ph ◽  
Ph Range ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of the hydrolysis of N,N-dimethyl-2-phenylaziridinium ion (DPA) have been studied over the pH range 5.5–8.0 as have the kinetics of the interaction of DPA with bovine erythrocyte acetyl-cholinesterase. The enzyme is initially inhibited reversibly and subsequently irreversibly towards acetylcholine hydrolysis. The hydrolysis of DPA was found to be pH independent over the range studied while the reversible noncompetitive inhibition increased with increasing pH, the data suggesting the requirement for a basic group on the enzyme with a pKa of about 6.5.Between pH values of 6.0 and 8.0 the kinetics of the irreversible inhibition are consistent with either of two kinetically indistinguishable mechanisms, one involving transformation of the initial reversible complex and the other an independent attack on the uncomplexed enzyme. The first mechanism gives rise to a first-order rate constant which is comparable with that for the hydrolysis of DPA but which increases with decreasing pH; an acidic group on the enzyme with pKa between 6.0 and 7.0 may be involved. The second-order rate constant arising from the second treatment goes through a maximum at pH 7.3. At pH 5.5 the kinetics are not consistent with either mechanism.

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