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.

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 Kinetics of Enzymatic Hydrolysis of Southeast Sulawesi Sago Starch

2020 ◽  
pp. 53-61
Author(s):  
Ansharullah Ansharullah ◽  
Muhammad Natsir
Keyword(s):  
Enzymatic Hydrolysis ◽  
Maximum Velocity ◽  
Enzyme Concentration ◽  
Hydrolysis Rate ◽  
Sago Starch ◽  
Optimum Conditions ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Menten Constant

The aims of this study were to characterize the kinetics of enzymatic hydrolysis of sago starch, obtained from Southeast Sulawesi Indonesia. The enzyme used for hydrolysis was bacterial ∝-amylase (Termamyl 120L from Bacillus licheniformis, E. C. 3.2.1.1).  The method to determine the initial velocity (Vo) of the hydrolysis was developed by differentiation a nonlinear equation (NLE).  The Vo of the hydrolysis was measured at various pH (6.0, 6.5,and 7.0), temperatures (40, 60, 75 and 95oC), enzyme concentrations (0.5, 1.0, 1.5 and 2.0 µg per mL) and in the presence of 70 ppm Ca++. The optimum conditions of this experiment were found to be at pH 6.5 – 7.0 and 75oC, and the Vo increased with increasing enzyme concentration. The Vo values at various substrate concentrations were also determined, which were then used to calculate the enzymes kinetics constant of the hydrolysis, including Michaelis-Menten constant (Km) and maximum velocity (Vmax) using a Hanes plot.  Km and Vmax values were found to be higher in the measurement at pH 7.0 and 75oC. The Km values  at four  different combinations of pH and temperatures (pH 6.5, 40oC; pH 6.5, 75oC; pH 7.0, 40oC; pH 7.0, 75oC) were found to be 0.86, 3.23, 0.77 and 3.83 mg/mL, respectively; and Vmax values were 17.5, 54.3, 20.3 and 57.1 µg/mL/min, respectively. The results obtained showed that hydrolysis rate of this starch was somewhat low.

Effects of gas composition and pH on kinetics of lung angiotensin-converting enzyme

1987 ◽  
Vol 62 (3) ◽  
pp. 1216-1221 ◽  
Author(s):  
D. A. Rickaby ◽  
R. D. Bongard ◽  
M. J. Tristani ◽  
J. H. Linehan ◽  
C. A. Dawson
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Surface Area ◽  
Ph Dependence ◽  
Gas Composition ◽  
Converting Enzyme ◽  
Hydrolysis Process ◽  
Hypoxic Vasoconstriction ◽  
Ace Activity ◽  
Kinetics Of ◽  
Hydrolysis Of

Given the pH dependence of enzymes in general and the potential importance of a blood and alveolar gas composition dependency on the interpretation of changes in the hydrolysis of angiotensin-converting enzyme (ACE) substrates by pulmonary endothelial ACE, we examined the influence of Pco2 and Po2 on the hydrolysis of a synthetic ACE substrate (benzoyl-phenylalanyl-alanyl-proline, BPAP) on passage through isolated rabbit lungs. Perfusate pH values of about 7.1, 7.4, and 7.9 were obtained by ventilating the lungs with gas containing different CO2 concentrations and Po2 values of approximately 110 and approximately 10 Torr were obtained by varying the concentration of O2 in the ventilating gas mixture. In the range studied neither acidosis nor alkalosis produced any significant changes in BPAP hydrolysis or in the kinetic parameters, Vmax and Km, for the hydrolysis process. On the other hand, a reduction in BPAP hydrolysis was detected when the Po2 was reduced from 110 to 10 Torr. The Vmax for BPAP hydrolysis by the lung was inversely correlated with the magnitude of the hypoxic vasoconstriction that occurred, suggesting that the reduced BPAP hydrolysis with hypoxia was due to the loss of perfused surface area due to the vasoconstriction. The results suggest that correlations between Pco2 and/or pH and whole-lung ACE activity that might occur in diseased lungs do not imply causalty. The hemodynamic consequences of changing Po2 (i.e., hypoxic vasoconstriction) may alter whole-organ ACE activity in the sense of changing the perfused surface area (i.e., the amount of ACE in contact with flowing perfusate).

KINETIC STUDIES ON THE HYDROLYSIS OF BENZOYLCHOLINE BY HUMAN SERUM CHOLINESTERASE

1956 ◽  
Vol 34 (1) ◽  
pp. 637-653 ◽  
Author(s):  
W. Kalow ◽  
K. Genest ◽  
N. Staron
Keyword(s):  
Kinetic Studies ◽  
Reaction Rates ◽  
Serum Cholinesterase ◽  
Initial Reaction ◽  
Ultraviolet Spectrophotometry ◽  
First Order ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

Benzoylcholine stands out from other known substrates of serum cholinesterase because of its high apparent affinity for this enzyme combined with a rapid rate of destruction. The reaction kinetics of the hydrolysis of benzoylcholine can be studied by ultraviolet spectrophotometry, since the absorbance decreases in proportion to the concentration of substrate. Kinetic data obtained by measuring initial reaction rates, and by analyzing continuous hydrolysis curves, are the same within the range of experimental error. The enzymatic data are compatible with the assumption that in the presence of high substrate concentrations a complex consisting of esterase and two substrate molecules is formed. This complex is hydrolyzed more slowly than the complex containing one molecule of substrate which is formed at low concentrations of benzoylcholine. Alkaline hydrolysis of benzoylcholine follows the kinetics of a first order reaction.

scholarly journals Direct Diels-Alder reaction of chitin derived 3-acetamido-5-acetylfuran

2022 ◽  
Author(s):  
Rafael Gomes ◽  
Juliana Pereira ◽  
João Ravasco ◽  
João Vale ◽  
Fausto Queda
Keyword(s):  
Alder Reaction ◽  
Diels Alder ◽  
Fine Tuning ◽  
Reduced Form ◽  
Partial Hydrolysis ◽  
Reaction Conditions ◽  
Diels Alder Reaction ◽  
Direct Use ◽  
Kinetics Of ◽  
Hydrolysis Of

The Diels-Alder (DA) reaction of biomass derived furans is an emerging technology for the preparation of new molecular entities and “drop-in” commodity chemicals. In this work we address the challenge of the direct use of electron-poor furanic platforms as dienes through the use of an unexplored chitin derived furan, 3-acetamido-5-acetylfuran (3A5AF). The 3-acetamido group promoted a remarkable increase in the kinetics of the DA allowing for the preparation of 7-oxanorbornenes (7-ONB) at 50 ºC. Partial hydrolysis of the enamide to hemi-acylaminals was possible upon fine tuning of the reaction conditions, disabling retro-DA processes. Finally, DA reaction of the reduced form of 3A5AF allowed quantitative formation of 7-ONB in aqueous condition after 10 minutes. Certanly these are the first steps for expanding the toolbox of chitin derived 3A5AF as diene.

Kinetics and mechanism of gold(III) incorporation into tetrakis(1-methylpyridium-4-yl)porphyrin in aqueous solution

2004 ◽  
Vol 08 (11) ◽  
pp. 1269-1275 ◽  
Author(s):  
Ahsan Habib ◽  
Masaaki Tabata ◽  
Ying Guang Wu
Keyword(s):  
Aqueous Solution ◽  
Rate Constants ◽  
Kinetic Data ◽  
Ph Dependence ◽  
Equilibrium Constants ◽  
Hydroxyl Group ◽  
Net Charge ◽  
First Order ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of the reaction of the tetrakis(1-methylpyridium-4-yl)porphyrin tetracation, [ H 2( TMPyP )]4+, with gold(III) ions were studied along with equilibria of gold(III) species in aqueous medium at 25°C, I = 0.10 M ( NaNO 3). The equilibrium constants for the formation of [ AuCl 4-n( OH ) n ]- ( n = 0,…,4), defined as β n = [ AuCl 4- n ( OH ) n ]- [ Cl -] n / [ AuCl 4-][ OH -] n were found to be that log β1 = 7.94 ± 0.03, log β2 = 15.14 ± 0.03, log β3 = 21.30 ± 0.05 and log β4 = 26.88 ± 0.05. The overall reaction was first order with respect to each of the total [ Au (III)] and [ H 2 TMPyP 4+]. On the basis of pH dependence on rate constants and the hydrolysis of gold(III), the rate expression can be written as d [ Au ( TMPyP )5+]/ dt = ( k 1[ AuCl 4-] + k2[ AuCl 3( OH )-] + k3[ AuCl 2( OH )2-] + k4[ AuCl ( OH )3-])[ H 2 TMPyP 4+], where k1, k2, k3 and k4 were found to be (2.16 ± 0.31) × 10-1, (6.56 ± 0.19) × 10-1, (1.07 ± 0.24) × 10-1, and (0.29 ± 0.21) × 10-1 M -1. s -1, respectively. The kinetic data revealed that the trichloromonohydroxogold(III) species, [ AuCl 3( OH )]-, is the most reactive. The higher reactivity of [ AuCl 3( OH )]- is explained by hydrogen bonding formation between the hydroxyl group of [ AuCl 3( OH )]- and the pyrrole hydrogen atom of [ H 2( TMPyP )]4+. Furthermore, applying the Fuoss equation to the observed rate constants at different ionic strengths, the apparent net charge of [ H 2( TMPyP )]4+ was calculated to be +3.5.

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.

scholarly journals DOES THE KINETICS OF TRYPSIN DIGESTION DEPEND ON THE FORMATION OF A COMPOUND BETWEEN ENZYME AND SUBSTRATE

1922 ◽  
Vol 4 (5) ◽  
pp. 487-509 ◽  
Author(s):  
John H. Northrop
Keyword(s):  
Experimental Evidence ◽  
Substrate Concentration ◽  
Relative Velocity ◽  
Mass Action ◽  
Law Of Mass Action ◽  
Rate Of Hydrolysis ◽  
Gelatin Concentration ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

1. The velocity of hydrolysis of gelatin by trypsin increases more slowly than the gelatin concentration and finally becomes nearly independent of the gelatin concentration. The relative velocity of hydrolysis of any two substrate concentrations is independent of the quantity of enzyme used to make the comparison. 2. The rate of hydrolysis is independent of the viscosity of the solution. 3. The percentage retardation of the rate of hydrolysis by inhibiting substances, is independent of the substrate concentration. 4. There is experimental evidence that the enzyme and inhibiting substance are combined to form a widely dissociated compound. 5. If the substrate were also combined with the enzyme, an increase in the substrate concentration should affect the equilibrium between the enzyme and the inhibiting substance. This is not the case. 6. The rate of digestion of a mixture of casein and gelatin is equal to the sum of the rates of hydrolysis of the two substances alone, as it should be if the rate is proportional to the concentration of free enzyme. This contradicts the saturation hypothesis. 7. If the reaction is followed by determining directly the change in the substrate concentration, it is found that this change agrees with the law of mass action; i.e., the rate of digestion is proportional to the substrate concentration.

Flow Kinetics of β-Galactosidase Chemically Attached to Nylon Tubing

1975 ◽  
Vol 53 (10) ◽  
pp. 1061-1069 ◽  
Author(s):  
D. Narinesingh ◽  
T. T. Ngo ◽  
K. J. Laidler
Keyword(s):  
Flow Rate ◽  
Substrate Concentration ◽  
Diffusion Control ◽  
Theoretical Treatment ◽  
Enzyme Reactors ◽  
Diffusion Controlled ◽  
Nylon Tubing ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

β-Galactosidase (EC 3.2.1.23) has been attached covalently to the inner surface of nylon tubing. An experimental study has been made of the flow kinetics for the hydrolysis of o-nitrophenylgalactose, the substrate concentration and flow rate being varied. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, three different methods of analysis being employed. It is concluded that at the lower substrate concentrations and flow rates employed, the reactions are largely diffusion controlled; with increase in flow rate and substrate concentration the width of the Nernst diffusion layer decreases, and there is found to be less diffusion control. The values of Km(app) vary with flow rate VF, being linear in VF−1/3, and the value extrapolated to very high flow rate agrees well with the Km value for β-galactosidase in free solution. The theory and results are shown to provide guidelines for the design of open tubular heterogeneous enzyme reactors for industrial, biomedical, and analytical applications.

scholarly journals Correlated biochemical and cytochemical studies of nicotinamide adenine dinucleotide phosphatase (NADPase) activity in ameloblasts using structural analogues of NADP.

1981 ◽  
Vol 29 (7) ◽  
pp. 822-836 ◽  
Author(s):  
C E Smith
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Phosphate Group ◽  
Nicotinamide Adenine ◽  
Reduced Form ◽  
Km Value ◽  
Covalently Linked ◽  
Hydrolysis Of ◽  
Menten Constant ◽  
Ethylene Group

The effects of altering the molecular structure of nicotinamide adenine dinucleotide phosphate (NADP) on enzymatic hydrolysis of the monoester phosphate group was examined at pH 5.0 in rat incisor ameloblasts using eight analogues of the oxidized form of the beta-isomer of NADP (beta-NADP+) modified in either the nicotinamide or adenine regions, or at the site for attachment of the monoester phosphate group to the molecule. Biochemical studies with whole homogenates of unfixed enamel organs revealed that the Michaelis-Menten constant (KM) and the maximum rate of dephosphorylation (Vmax) were different for these analogues relative to values estimated with beta-NADP+ as substrate. For example, the KM value was 3-fold higher and the Vmax value was lower by about 1/2 with the reduced form of the molecule as substrate. The KM value was about 2-fold higher but the Vmax value was about the same with an analogue lacking the nicotinamide group as substrate, while both the KM and the Vmax values were about 2-fold higher with an analogue containing an ethylene group covalently linked to adenine as substrate. In contrast, the KM value was markedly reduced (1/15) and the Vmax value was elevated (4-fold) using an analogue as substrate which contained the phosphate group attached at the 3'-position (3'-NADP+), rather than the 2'-position as in beta-NADP+ and other analogues. Cytochemical studies with glutaraldehyde-fixed enamel organs revealed that reaction product from hydrolysis of beta-NADP+ and other analogues containing the phosphate group attached at the natural 2'-position was localized within the intermediate saccules of the ameloblast Golgi apparatus. Reaction product from hydrolysis of 3'-NADP+, however, was localized at a different site, that is, within granules and membranous connections of the GERL system in the cell. Hence, 3'-NADP+ was not hydrolyzed by NADPase but by another enzyme tentatively identified as Coenzyme A phosphatase.

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