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K J Laidler

Publications and source records attributed to K J Laidler.

14 recordsLinked to original sources
PubMed1986

Flow kinetics of immobilized beta-glucosidase.

The enzyme beta-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 degrees 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 degrees 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.

Calorimetry↗
PubMed1979

The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching.

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.

Absorption↗
PubMed1979

Kinetics of acetylcholinesterase immobilized on polyethylene tubing.

Acetylcholinesterase was covalently attached to the inner surface of polyethylene tubing. Initial oxidation generated surface carboxylic groups which, on reaction with thionyl chloride, produced acid chloride groups; these were caused to react with excess ethylenediamine. The amino groups on the surface were linked to glutaraldehyde, and acetylcholinesterase was then attached to the surface. Various kinetic tests showed the catalysis of the hydrolysis of acetylthiocholine iodide to be diffusion controlled. The apparent Michaelis constants were strongly dependent on flow rate and were much larger than the value for the free enzyme. Rate measurements over the temperature range 6-42 degrees C showed changes in activation energies consistent with diffusion control.

Acetylcholinesterase↗
PubMed1978

Temperature and pH effects with immobilized electric eel acetylcholinesterase.

Kinetic studies were made with 2 forms of immobilized acetylcholinesterase: enzyme trapped in polyacrylamide gel which was cut into slices; and enzyme attached to the inner surface of nylon tubing. Rates were measured at substrate concentrations which were low and high with reference to the Michaelis constant, and over the temperature range 16-40 degrees C. Low activation energies (1.7-2.7 kcal mol-1) were obtained at low substrate concentrations, indicating diffusion control. At high substrate concentrations the Arrhenius plots were non-linear and the activation energies substantially higher, and there is less diffusion control. With enzyme-polyacrylamide slices, there was a continuous increase in rate with increasing pH, in contrast to the bell-shaped behavior with free enzyme. A theoretical treatment suggests that this is due to the lowering of local pH as a result of the acid released in the hydrolysis.

Acetylcholinesterase↗
PubMed1978

Flow kinetics of lactate dehydrogenase chemically attached to nylon tubing.

Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing; a modified technique, involving benzidine and glutaraldehyde, was used, and the resulting immobilized enzyme showed no loss of activity over a period of several months. An experimental study was made of the flow kinetics for the reaction between pyruvate and reduced nicotinamide adenine dinucleotide in two limiting cases, one substrate in excess and the concentration of the other one varied. A range of flow rates and temperatures was covered. The results were analyzed in various ways on the basis of the Kobayashi--Laidler treatment of flow systems. It was concluded that the kinetics are largely diffusion-controlled, especially at the lower substrate concentrations and flow rates. The values of the apparent Michaelis constants vary with flow rate vf, being linear in vf-1/3, and the values extrapolated to infinite flow rate (vf-1/3 = 0) approach the values for the enzyme in free solution. Analysis of the rates led to activation energies for the diffusion of the two substrates.

Enzymes, Immobilized↗
PubMed1976

Hydrolysis of D-galactosides in an open tubular lactase reactor.

Lactase (beta-galactosidase) was attached to the inner surface of nylon tubing. Tubes of various lengths were used to bring about the hydrolysis of o-nitrophenyl-beta-D-galactoside and of lactose in skim milk. The results with the former substrate were analyzed in the light of a theoretical treatment of Kobayashi and Laidler (Biotechnol. Bioeng., 16, 99, 1974), with the conclusion that the reaction is intermediate between diffusion-free and completely diffusion-controlled behavior. The results with skim milk show that with a single 46 m tube and continuous circulation, 90% of the lactose is removed within 20 hr. A battery of ten such tubes, with single passage, at a flow rate of 2 cm/sec, would remove more than 99% of the lactose in less than 40 min.

Animals↗
PubMed1976

Four-and five-step kinetic models of lactate dehydrogenase.

A five-step model for the reaction catalyzed by beef heart lactate dehydrogenase (EC 1.1.1.27) reconciles differences observed in the four-step model if pre-steady-state data in the forward direction are compared with similar data in the reverse direction. The relationship between the four-and five-step models indicates what problems can develop when an incomplete model is proposed. Nevertheless, there are advantages to using the less complicated four-step model when comparing the molecular kinetics of enzymes catalyzing the same reaction but obtained from different sources.

Animals↗
PubMed1975

Immobilized electric eel acetylcholinesterase. I. Kinetics of acetylcholinesterase trapped in polyacrylamide membranes.

Techniques are described for the trapping of electric eel acetylcholinesterase in polyacrylamide gel. The activity of the trapped enzyme was substantially reduced, the effect being due to inhibition by acrylamide, but the emzyme immobilized in polyacrylamide was considerable more stable than that in free solutionma kinetic study was made of the hydrolysis of acetylthiocholine, covering a range of membrane thicknesses, enzyme concentrations, substrate concentrations and temperatures. The results were interpreted with reference to the theoretical treatment of Sundaram, Tweedale and Laidler, and of Kobayaski and Laidler, and provided support for those treatments; Clear evidence was obtained for diffusion control with the thicker membranes. An activation energy was obtained for the diffusion of the substrate within the membrane, by combining the temperature results for thick and thin membranes at low substrate concentrations. The results lead to the conclusion that the in vivo kinetics of acetylcholinesterase are largely diffusion-free in muscle filaments, but are substantially diffusion-controlled in fibrils and fibers.

Acetylcholinesterase↗
PubMed1975

Immobilized electric eel acetylcholinesterasemii. II. Flow kinetics of acetylcholinesterase chemically attached to nylon tubing.

Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes.

Acetylcholinesterase↗
PubMed1975

A flow method for determination of the kinetic parameters for immobilized enzymes.

A flow method is described for determination of the kinetics parameters (V-m and K-m) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate of formation of product. To illustrate the technique, it is applied to the determination of the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing.

Acetylcholinesterase↗
PubMed1975

Transient kinetics of the acetylcholinesterase catalyzed hydrolysis of N-methylindoxyl acetate.

An experimental study has been made of the kinetics of the hydrolysis of N-methylindoxyl acetate catalyzed by electric-eel acetylcholinesterase, both in the steady state and the pre-steady statemstopped-flow and temperature-jump experiments revealed a fast transient and a slow one. The fast transient is correlated with the conventional mechanism E+A in equilibrium EA yields X-yieldsEA' YIELDS E+Y. The slow transient is attributed to conformational changes involving E or EA. Analysis of it revealed two exponential terms of the form e- minus lambda t, and the two lambda values were obtained over the temperature range 5Yand 25Ydegrees C. The results are interpreted in terms of two alternative mechanisms; in one, the enzyme undergoes a conformational change before it adds on the substrate molecule; in the other, the conformational change occurs after the substrate addition. Both mechanisms may be involved, but the results exclude a concerted mechanism in whivh the conformational change occurs concurrently with the addition of substrate. Kinetic parameters (delta S not equal to and E) are obtained for this conformational change and for the conversion of EA into EA'+X.

Acetates↗
PubMed1975

Flow kinetics of beta-galactosidase chemically attached to nylon tubing.

Beta-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 beta-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.

Diffusion↗
PubMed1975

Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.

Animals↗