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 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.

CHARACTERIZATION OF THREE HEMICELLULOSES FROM THE WOOD OF AMABILIS FIR (ABIES AMABILIS)

1963 ◽  
Vol 41 (6) ◽  
pp. 1381-1388 ◽  
Author(s):  
E. C. A. Schwarz ◽  
T. E. Timell
Keyword(s):  
Periodate Oxidation ◽  
Specific Rotation ◽  
Water Soluble ◽  
Partial Hydrolysis ◽  
Abies Amabilis ◽  
A Value ◽  
Linear Framework ◽  
Molecular Rotations ◽  
Hydrolysis Of

An arabino-4-O-methylglucuronoxylan (10:14:76), a water-soluble galactoglucomannan (1:1:3), and an alkali-soluble galactoglucomannan (0.1:1:3) have been isolated in yields of 7, 4, and 8% from the wood of amabilis fir (Abies amabilis). The structure of the polysaccharides was established by methylation and periodate oxidation techniques. The xylan ([Formula: see text]) contained side chains of (1 → 2)-linked 4-O-methyl-α-D-glucuronic acid and (1 → 3)-linked α-L-arabinofuranose residues, both attached directly to a linear framework of (1 → 4)-linked β-D-xylose residues. The galactoglucomannans ([Formula: see text] 76 and 95) contained (1 → 6)-linked α-D-galactopyranosyl residues attached directly to a backbone of (1 → 4)-linked β-D-mannose and β-D-glucose residues. Partial hydrolysis of the two hexosans yielded 10 and 13 oligosaccharides, respectively, containing (1 → 4)-β-D-mannose, (1 → 4)-β-D-glucose, and (1 → 6)-α-D-galactopyranose residues. The molecular rotations of a series of mannooligosaccharides corresponded to a value of −54° for the specific rotation of an infinitely long mannan. It is concluded that the main hemicelluloses in amabilis fir wood are the same as those occurring in the wood of gymnosperms from other genera. The same three hemicelluloses are also present in the bark of this species.

Flow kinetics of immobilized β-glucosidase

1986 ◽  
Vol 64 (2) ◽  
pp. 139-145 ◽  
Author(s):  
Yuchiong Hsuanyu ◽  
Keith J. Laidler
Keyword(s):  
Ph Dependence ◽  
Kinetic Studies ◽  
Free Enzyme ◽  
Diffusion Control ◽  
Michaelis Constant ◽  
Ph Optimum ◽  
Ph Values ◽  
Nylon Tubing ◽  
Kinetics Of ◽  
Substrate Concentrations

The enzyme β-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 °C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 °C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.

Rates of hydrolysis of a monochlorotriazine dye in presence of non-ionic surfactant

1968 ◽  
Vol 21 (4) ◽  
pp. 1007 ◽  
Author(s):  
BR Craven ◽  
A Datyner ◽  
TP Doyle
Keyword(s):  
Complex Formation ◽  
Ethylene Oxide ◽  
Rate Constant ◽  
The Other ◽  
Ionic Surfactant ◽  
A Value ◽  
Surfactant Complex ◽  
Rate Of Hydrolysis ◽  
Ethylene Oxide Chain ◽  
Hydrolysis Of

The presence of nonylphenol-poly(oxyethylene) surfactant decreases the rate constant for the hydrolysis of a monochlorotriazine dye, the effect being enhanced by increasing the surfactant to dye ratio and diminishing by increasing the temperature. At 80� and 100�, the longer the ethylene oxide chain length of a surfactant the more effective it is in reducing the rate constant. At 60�, however, all of the surfactants used appear to be equally effective. At 80� and 100�, extrapolation of rate constants to zero surfactant to dye ratio gives a value very close to the rate constant in the absence of surfactant, but at 60� the rate constant for NPl0 extrapolated to zero surfactant to dye ratio is close to the value from the surfactant-free run, but values obtained with the other surfactants are much higher and similar to that obtained in presence of a polyether glycol. It is suggested that this anomaly is due to the initial aggregation of the dye at 60� so that the addition of small quantities of surfactant other than NPl0 causes initial disaggregation and an increase in the rate of hydrolysis. Further increases in the amount of surfactant cause dye-surfactant complex formation which produces a decrease in the rate of hydrolysis.

The mutation of Thr315 to Asn of GH10 xylanase XynR increases the alkaliphily but decreases the alkaline resistance

2021 ◽  
Author(s):  
Kohei Kuwata ◽  
Manami Suzuki ◽  
Teisuke Takita ◽  
Rie Yatsunami ◽  
Satoshi Nakamura ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Culture Broth ◽  
Saturation Mutagenesis ◽  
Ph Values ◽  
Beechwood Xylan ◽  
Alkaline Resistance ◽  
Dependent Activity ◽  
A Site ◽  
Hydrolysis Of ◽  
Gh10 Xylanase

Abstract XynR is a thermophilic and alkaline GH10 xylanase, identified in the culture broth of alkaliphilic and thermophilic Bacillus sp. strain TAR-1. We previously selected S92E as a thermostable variant from a site saturation mutagenesis library. Here, we attempted to select the alkaliphilic XynR variant from the library and isolated T315N. In the hydrolysis of beechwood xylan, T315N and S92E/T315N exhibited a broader bell-shaped pH-dependent activity than the wild-type XynR (WT) and S92E. The optimal pH values of T315N and S92E/T315N were 6.5–9.5 while those of WT and S92E were 6.5–8.5. On the other hand, T315N and S92E/T315N exhibited a narrower bell-shaped pH-dependence of stability: the pHs at which the activity was stable after the incubation at 37 °C for 24 h were 6.0–8.5 for T315N and S92E/T315N, but 6.0–10.0 for WT and S92E. These results indicated that the mutation of Thr315 to Asn increased the alkaliphily but decreased the alkaline resistance.

Kinetics of the hydrolysis of cellobiose catalysed by β-glucosidase

1985 ◽  
Vol 63 (3) ◽  
pp. 167-175 ◽  
Author(s):  
Yuchiong Hsuanyu ◽  
Keith J. Laidler
Keyword(s):  
Ph Dependence ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reduced Form ◽  
Arrhenius Behavior ◽  
Elementary Reactions ◽  
Individual Reaction ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

The hydrolysis of cellobiose catalysed by β-glucosidase has been investigated by experimental techniques which allow the course of reaction to be followed continuously. They involve assaying the product glucose by the use of ATP, hexokinase, glucose-6-phosphate dehydrogenase, and nicotinamide adenine dinucleotide phosphate (NADP); the latter is converted into its reduced form NADPH which absorbs strongly at 340 nm. Rates were measured at nine pH values varying from 5 to 6.9, at substrate concentrations varying from 0.2 to 3.2 mM, and at temperatures varying from 10 to 37 °C. The pH dependence revealed pK values of 4.9 and 6.5 in the free enzyme at 24 °C, and these are little changed on complex formation. The rates measured over a range of temperature, as interpreted by Arrhenius plots, revealed an inflexion at 23 °C, found consistently under all conditions. The results are analyzed in terms of the mechanism[Formula: see text]It was found possible to obtain, for the four elementary reactions, activation energies and entropies of activation which explain the inflexion at 23 °C and the Arrhenius behavior above and below that temperature. Profiles are constructed showing the variations of entropy and enthalpy during the course of an individual reaction.

Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

2019 ◽  
Author(s):  
Adrian Roitberg ◽  
Pancham Lal Gupta
Keyword(s):  
Transfer Reaction ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
Ph Effects ◽  
Dependent Manner ◽  
E Coli ◽  
Gar Tfase ◽  
Activity Curve ◽  
Ph Dependent ◽  
Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

scholarly journals β-Glucosidase Activity of Lactiplantibacillus plantarum UNQLp 11 in Different Malolactic Fermentations Conditions: Effect of pH and Ethanol Content

Fermentation ◽  
2021 ◽  
Vol 7 (1) ◽  
pp. 22
Author(s):  
Natalia S. Brizuela ◽  
Marina Arnez-Arancibia ◽  
Liliana Semorile ◽  
María Ángeles Pozo-Bayón ◽  
Bárbara M. Bravo-Ferrada ◽  
...  
Keyword(s):  
Pinot Noir ◽  
Gas Chromatography Mass Spectrometry ◽  
Red Wines ◽  
Ph Values ◽  
Glucosidase Activity ◽  
Sorptive Extraction ◽  
Ethanol Content ◽  
Volatile Precursors ◽  
Hydrolysis Of ◽  
High Ethanol

Lactiplantibacillus plantarum strain UNQLp 11 is a lactic acid bacterium with the potential to carry out malolactic fermentation (MLF) in red wines. Recently, the complete genome of UNQLp 11 was sequenced and this strain possesses four loci of the enzyme β-glucosidase. In order to demonstrate that these glucosidase enzymes could be functional under harsh wine conditions, we evaluated the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (p-NPG) in synthetic wine with different ethanol contents (0%, 12%, and 14% v/v) and at different pH values (3.2, 3.5, and 3.8). Then, the hydrolysis of precursor n-octyl β-D-glucopyranoside was analyzed in sterile Pinot Noir wine (containing 14.5% v/v of ethanol, at different pH values) by headspace sorptive extraction gas chromatography-mass spectrometry (HSSE-GC/MS). The hydrolysis of p-NPG showed that β-glucosidase activity is very susceptible to low pH but induced in the presence of high ethanol content. Furthermore, UNQLp 11 was able to release the glycosilated precursor n-octyl, during MLF to a greater extent than a commercial enzyme. In conclusion, UNQLp 11 could improve the aromatic profile of the wine by the release of volatile precursors during MLF.

Preparation and structural analysis of (±)-threo-ritalinic acid

2013 ◽  
Vol 69 (11) ◽  
pp. 1225-1228 ◽  
Author(s):  
Sara Wyss ◽  
Irmgard A. Werner ◽  
W. Bernd Schweizer ◽  
Simon M. Ametamey ◽  
Selena Milicevic Sephton
Keyword(s):  
Methyl Ester ◽  
Free Acid ◽  
Nmr Analysis ◽  
Intermolecular Hydrogen Bonds ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ph Values ◽  
Ritalinic Acid ◽  
Methyl Phenidate ◽  
Hydrolysis Of

Hydrolysis of the methyl ester (±)-threo-methyl phenidate afforded the free acid in 40% yield,viz.(±)-threo-ritalinic acid, C13H17NO2. Hydrolysis and subsequent crystallization were accomplished at pH values between 5 and 7 to yield colourless prisms which were analysed by X-ray crystallography. Crystals of (±)-threo-ritalinic acid belong to theP21/nspace group and form intermolecular hydrogen bonds. An antiperiplanar disposition of the H atoms of the (HOOC—)CH—CHpygroup (py is pyridine) was found in both the solid (diffraction analysis) and solution state (NMR analysis). It was also determined that (±)-threo-ritalinic acid conforms to the minimization of negativegauche+–gauche−interactions.

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