scholarly journals The apparent Km is a misleading kinetic indicator

1986 ◽  
Vol 239 (1) ◽  
pp. 175-178 ◽  
Author(s):  
I W Plesner
Keyword(s):  
Substrate Concentration ◽  
Kinetic Data ◽  
Substrate Binding ◽  
Enzymic Activity ◽  
Free Enzyme ◽  
Ligand Concentration ◽  
Double Reciprocal Plot ◽  
Michaelis Constant ◽  
Formal Framework ◽  
Enzyme Species

When information concerning whether or not a ligand interacts with the same enzyme species as do the substrates, the variation of the Michaelis constant Km (for each substrate) with ligand concentration is sometimes used as a diagnostic. It is shown that the Michaelis constant is of no particular value in this respect and may be misleading. Thus, depending on the mechanism, Km may vary with ligand concentration even though the ligand interacts with species far removed in the mechanism from the substrate-binding steps, and it may stay constant in cases where the ligand competes directly for the free enzyme. In contrast, the slope of a double-reciprocal plot of the kinetic data (= Km/Vmax.) (or, equivalently, the ordinate intercept of a Hanes plot A/v versus A, where A is the substrate concentration) independently of the particular mechanism involved uniquely signifies whether or not such interaction occurs. The results clearly indicate that, for purposes other than communicating the substrate concentration yielding control of the enzymic activity, usage of Km and its variation with ligand concentration should be avoided and interest instead focused on the slope, in accordance with the long-established rules of Cleland [Biochim. Biophys. Acta (1963) 67, 188-196], for which the present analysis provides the formal framework.

β-GLUCURONIDASE OF RABBIT POLYMORPHONUCLEAR LEUCOCYTES

1950 ◽  
Vol 28e (3) ◽  
pp. 69-79 ◽  
Author(s):  
R. J. Rossiter ◽  
Esther Wong
Keyword(s):  
Enzyme Activity ◽  
Blood Plasma ◽  
Substrate Concentration ◽  
Final Concentration ◽  
White Cell ◽  
Polymorphonuclear Leucocytes ◽  
Michaelis Constant ◽  
Glucuronidase Activity ◽  
Arsanilic Acid ◽  
Optimum Ph

Rabbit polymorphonuclear leucocytes contain an enzyme capable of hydrolyzing biosynthetic phenolphthalein mono-β-glucuronide. The concentration of the enzyme in the white cell is some 2000 times the concentration of the enzyme in the blood plasma. Under the conditions of study, the β-glucuronidase activity was proportional to the concentration of the enzyme. The effect of substrate concentration on the enzyme activity was studied and the Michaelis constant, Ks, determined. The course of the reaction was linear with time for the first 12 hr. and then fell off slightly during the next 12 hr. The optimum pH of the enzyme was 4.45 in either 0.2 M acetate or 0.2 M phthalate buffer. It was not inhibited by cyanide, azide, iodoacetate, fluoride, glycine, thiourea, urethane, arsanilic acid, acetophenone, o-cresol or m-cresol, in a final concentration of 0.01 M. The possible function of β-glucuronidase in rabbit polymorphonuclear leucocytes is discussed.

scholarly journals Graphical Approach to Compare Concentration Constants of Hill and Michaelis-Menten Equations

2018 ◽  
Vol 1 (3) ◽  
pp. 94-99 ◽  
Author(s):  
Elena V. Emelyanova
Keyword(s):  
Catalytic Activity ◽  
Substrate Concentration ◽  
Hill Equation ◽  
Enzymatic Process ◽  
Michaelis Constant ◽  
Graphical Approach ◽  
Graphical Technique ◽  
Kinetics Of ◽  
Permissible Rate ◽  
Concentration Constants

The aim of present study was to describe the graphical technique how to go from Hill concentration constant to Michaelis constant. To compare enzymatic processes, the kinetics of which is subjected to different regularities, it is possible to use constants that characterize catalytic activity (Vmax) and concentration constants that are the substrate concentration at which the rate of the enzymatic process is equal to a half of maximum permissible rate. Concentration constants are S0.5 for Hill equation and Km for Michaelis-Menton equation. The graphical approach was proposed in order to go from concentration constant of Hill equation to Michaelis concentration of the process that could be characterized by the same catalytic activity (the same values of minimum and maximum rates) similar to that observed in the process described by Hill equation.

The effect of increasing substrate concentration on the gas production profiles of pure maize starch and milled, high temperature dried grass

1996 ◽  
Vol 1996 ◽  
pp. 222-222
Author(s):  
J. A. Huntington ◽  
D. I. Givens
Keyword(s):  
Substrate Concentration ◽  
Linear Response ◽  
Pressure Transducer ◽  
Rumen Fluid ◽  
Lag Time ◽  
Enzymic Activity ◽  
Gas Production ◽  
Maize Starch ◽  
Data Logger ◽  
Manual Pressure

Theodorou et al. (1994) using a manual pressure transducer technique observed a linear response to substrate concentration (cone.) in gas production (GP; ml absolute) whilst lag time and rate of GP values remained constant. This experiment investigated the effect of increasing the cone, of maize starch (S) and milled (1mm) dried grass (DG) on the GP profile using an automated GP technique (Cone 1994).Either 0.1, 0.25, 0.5, 1.0 or 2.0 g of S or DG were weighed into 250 ml bottles and pre-soaked with anaerobic buffer (85 ml). The bottles were connected to a data logger/pressure tranducer unit and gassed with carbon dioxide. Incubation (39°C) started ∽16 h later when strained rumen fluid was injected into each bottle (15 ml) and a data logger initiated. When GP had ceased the final pH of each bottle was measured and 25 ml of 1M H3PO4 added to stop further enzymic activity. DG bottles were filtered to determine organic matter (OM) residue and hence OM degraded (OMD).

The reactions of O(3P) with the butanols

1983 ◽  
Vol 61 (12) ◽  
pp. 2716-2720 ◽  
Author(s):  
John M. Roscoe
Keyword(s):  
Free Energy ◽  
Gas Phase ◽  
Rate Constants ◽  
Substrate Concentration ◽  
Kinetic Data ◽  
Absolute Rate ◽  
Gas Phase Reactions ◽  
Linear Free Energy ◽  
Different Types ◽  
Energy Relations

The reactions of O(3P) with the butanols were studied kinetically as a function of temperature and substrate concentration. The absolute rate constants for the gas phase reactions, in the units M−1 s−1, obey the following relations.[Formula: see text]The results suggest that although the α-CH bond in these alcohols is the most reactive one, reaction of O(3P) with other CH bonds in the alcohols is also appreciable. The kinetic data for these and other alcohols are separated into contributions from the different types of CH bonds and the results are discussed in terms of linear free energy relations.

Kinetic laws for solid-supported enzymes

1970 ◽  
Vol 48 (10) ◽  
pp. 1498-1504 ◽  
Author(s):  
P. V. Sundaram ◽  
A. Tweedale ◽  
K. J. Laidler
Keyword(s):  
Substrate Concentration ◽  
Free Solution ◽  
Kinetic Behavior ◽  
Michaelis Constant ◽  
Solid Supports ◽  
High Substrate ◽  
Substrate Concentrations

Enzymes behave differently when attached to solid supports for four main reasons: (1) their conformations when they are supported may differ from those in free solution, (2) they act upon substrates in a different environment, (3) there will be partitioning of substrate between the support and the free solution, and (4) there will be effects due to diffusion of the substrate in the support. The present paper examines effects (3) and (4) and shows how rates will vary with substrate concentration. If factors (1) and (2) do not enter, rates in the limit of high substrate concentrations will be the same for the supported enzyme as in free solution. At low substrate concentrations, rates will be less for the supported enzyme if the substrate is less soluble in the support than in free solution, and the apparent Michaelis constant, Km(app.), will be greater; conversely, for higher solubility in the support, rates will be greater and Km(app.) smaller. Effect (4) leads to lower rates and higher Km(app.) values, except in the limit of high substrate concentrations. At a sufficiently low thickness of the support, depending upon the activity of the enzyme, the kinetic behavior becomes identical with that in free solution.

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 Models for enzyme superactivity in aqueous solutions of surfactants

1999 ◽  
Vol 344 (3) ◽  
pp. 765-773 ◽  
Author(s):  
Paolo VIPARELLI ◽  
Francesco ALFANI ◽  
Maria CANTARELLA
Keyword(s):  
Substrate Concentration ◽  
Surfactant Concentration ◽  
Reaction Rate ◽  
Reaction Medium ◽  
Bound Water ◽  
Free Water ◽  
Theoretical Models ◽  
Enzymic Activity ◽  
Equilibrium Relation ◽  
Micellar Aggregates

Theoretical models are developed here for enzymic activity in the presence of direct micellar aggregates. An approach similar to that of Bru et al. [Bru, Sánchez-Ferrer and Garcia-Carmona (1989) Biochem. J. 259, 355-361] for reverse micelles has been adopted. The system is considered to consist of three pseudo-phases: free water, bound water and surfactant tails. The substrate concentration in each pseudo-phase is related to the total substrate concentration in the reaction medium. In the absence of interactions between the enzyme and the micelles, the model predicts either monotonically increasing or monotonically decreasing trends in the calculated reaction rate as a function of surfactant concentration. With enzyme-micelle interactions included in the formulation (by introducing an equilibrium relation between the enzyme confined in the free water and in the bound water pseudo-phases, and by allowing for different catalytic behaviours for the two forms), the calculated reaction rate can exhibit a bell-shaped dependence on surfactant concentration. The effect of the partition of enzyme and substrate is described, as is that of enzyme efficiency in the various pseudo-phases.

scholarly journals Purification and characterization of α-l-fucosidase from the liver of a fucosidosis patient

1980 ◽  
Vol 187 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Jack A. Alhadeff ◽  
Grai L. Andrews-Smith
Keyword(s):  
Immunoglobulin G ◽  
Normal Liver ◽  
Maximal Activity ◽  
Enzymic Activity ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Activity Curve ◽  
Normal Enzyme ◽  
Acid Region

α-l-Fucosidase was partially purified from the liver of a fucosidosis patient by column chromatography either on agarose–ε-aminohexanoylfucosamine or on concanavalin A–Sepharose, despite no apparent enzymic activity in the crude liver supernatant. Mixing studies indicated that the liver of the fucosidosis patient did not lack activators or contain inhibitors of α-l-fucosidase activity. The partially purified α-l-fucosidase from the liver of the fucosidosis patient exhibits a 4–5-fold-increased Michaelis constant for the 4-methylumbelliferyl substrate (700–750μm) and a greatly decreased thermostability at 55°C compared with the normal liver enzyme. The pH–activity curve is similar to that for the normal enzyme between pH5 and 8, but quite dissimilar in the acid region (pH3.0–4.5): below pH4.5 the α-l-fucosidase shows no activity, whereas the normal enzyme retains considerable activity (≥50% of maximal activity). Isoelectric focusing of the α-l-fucosidase revealed one major form with pI5.8 and other possible minor forms. No cross-reacting material was detected when the α-l-fucosidase was run in double-immunodiffusion experiments against the immunoglobulin-G fraction of anti-(α-l-fucosidase) antibodies, but the enzyme was immunoprecipitated by this immunoglobulin-G fraction. For at least the fucosidosis patient being studied here, all the data suggest retention of a thermolabile portion of normal α-l-fucosidase (with characteristic Michaelis constant and pH–activity curve) or production of a kinetically altered α-l-fucosidase with decreased catalytic activity but antigenic similarity to the normal enzyme.

scholarly journals Co-operativity and the methods of plotting binding and steady-state kinetic data

1978 ◽  
Vol 171 (2) ◽  
pp. 501-504 ◽  
Author(s):  
E P Whitehead
Keyword(s):  
Steady State ◽  
Ligand Binding ◽  
Substrate Concentration ◽  
Kinetic Data ◽  
Free Ligand ◽  
Free Energies ◽  
Steady State Kinetic ◽  
Second Derivatives ◽  
The Hill ◽  
State Kinetic

The features that distinguish positive from negative co-operativity in double-reciprocal Eadie-Hofstee-Scatchard and Hanes plots, often incorrectly stated to be the sign of curvature or second derivatives, are explained. It is shown how to determine the ‘Hill exponent’ and interaciton free energies from curves in these plots, and in the simple plot of ligand binding or velocity against free ligand or substrate concentration. New types of plots, where the kind of co-operative behaviour is more obvious than in the traditional ones, are proposed.

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