Staphylococcal L-asparaginase: enzyme kinetics.

The ph optimum of purified staphylococcal L-asparaginase (EC 3.5.1.1) was found to be between 8.6 and 8.8. The temperature optimum was 30 degrees-32 degrees C and the highest reaction rate occurred at 30 degrees C. The KM of the enzyme calculated from Lineweaver-Burk plot was 3.71 x 10(-2) M. Besides L-asparaginase, the substrate specificity of enzyme was restricted to N-alpha-acetyl-L-asparagine. D-asparagine, L-aspartic acid and D-glutamic acid were competitive inhibitors. Hg2+ and Cu2+ cations strongly inhibited the enzyme while Na+ and K+ cations strongly stimulated activity. Two SH-groups could be detected after enzyme denaturation with guanidine.     

A microscopic model of enzyme kinetics.

Many in vivo enzymatic processes, such as those of the tissue factor pathway of blood coagulation, occur in environments with facilitated substrate delivery or enzymes bound to cellular or lipid surfaces, which are quite different from the ideal fluid environment for which the Michaelis-Menten equation was derived. To describe the kinetics of such reactions, we propose a microscopic model that focuses on the kinetics of a single-enzyme molecule. This model provides the foundation for macroscopic models of the system kinetics of reactions occurring in both ideal and nonideal environments. For ideal reaction systems, the corresponding macroscopic models thus derived are consistent with the Michaelis-Menten equation. It is shown that the apparent Km is in fact a function of the mechanism of substrate delivery and should be interpreted as the substrate level at which the enzyme vacancy time equals the residence time of ES-complexes; it is suggested that our microscopic model parameters characterize more accurately an enzyme and its catalytic efficiency than does the classical Km. This model can also be incorporated into computer simulations of more complex reactions as an alternative to explicit analytical formulation of a macroscopic model.     

High pressure enzyme kinetics of dextransucrase.

The pressure dependence of enzymatic dextran formation has been observed up to 1000 at for several substrate concentrations. First order denaturation effects could be separated from the thermodynamic effects, which lead to a volume of 30.4 to 44.0 ccm per mole for the formation and -13.6ccm per mole for the activation of the enzyme-substrate complex. Denaturation depends on the substrate concentration. This leads to the conslusion that only the free enzyme is denatured, wheras the ES complex is stable.     

Convenient method for studying enzyme kinetics.

A convenient method for enzyme kinetic studies is introduced. The method includes identification of reaction mechanism and estimation of the associated kinetic constants with a minimum number of experiments. The application of the method is illustrated by using literature data. Factors limiting the application of this method are also discussed.     

Schematic methods for probabilistic enzyme kinetics.

The theory of steady-state enzyme processes which avoids using the mass action law of chemical kinetics and consistently describes catalytic mechanisms by probabilistic concepts has recently been proposed (Mazur, 1991, J. theor. Biol. 148, 229-242). To facilitate the analysis of complex reaction graphs by this theory the possibility of constructing schematic rules similar to those used in classical kinetics is studied. It is found that due to the similarity of algebraic procedures the popular method of King & Altman can be applied in probabilistic kinetics in addition to the earlier proposed rule based on enumeration of cycles of the reaction graph. This similarity also allows one to adapt many other shortcut methods of classical kinetics for probabilistic reaction graphs. The paper considers separately the possibility of transforming reaction mechanisms so that the initial graph is replaced by a simpler but equivalent one. It is shown that there are few cases when a group of states can be replaced by one united state, with earlier known rules such as the rule of Cha for equilibrium stages being particular cases of a more general procedure. In addition a novel method is proposed which performs step-by-step reduction of any reaction graph. All the new methods can be adapted for traditional kinetics as well. The results obtained demonstrate that many schematic rules of classical kinetics are of probabilistic origin.     

Application of topological methods in enzyme kinetics.

A criticism [Cornish-Bowden (1976) Biochem. J. 159, 167] of an algebraic method for deriving steady-state rate equations [Indge & Childs (1976) Biochem. J. 155, 567-570] is theoretically founded.     

The computation of hyperbolic dependences in enzyme kinetics.

A computer program aimed at analysing results following Michaelis-Menten kinetics can be used unmodified in the treatment of other kinetic results provided that the kinetic equations in these cases can be written in the form of the Michaelis-Menten equation. A list is presented of the parameters to be set instead of substrate concentration and reaction rate, and of constants replacing Km and V, if such a program is applied in analysing enzyme inhibitions, activations and pH-dependences.     

Prostaglandin H synthetase as a multisubstrate enzyme. Fluorimetric study of enzyme kinetics

The kinetics of a multisubstrate enzymatic reaction catalyzed by prostaglandin H synthase (PGH-synthase, EC 1.14.99.1) was studied, using homovanillic acid, a new electron donor for the given system. Homovanillic acid was shown to be a participant in a reaction with arachidonic acid/O2 stoichiometric ratios and is oxidized to a readily fluorescing product with an absorbance maximum (excitation) at 315 nm and fluorescence maximum at 425 nm. This allows for determination of the rate of enzymatic reaction with the sensitivity exceeding by one order of magnitude that of polarographic or spectrophotometric assays. Using fluorescent techniques, the dependence of the rate of PGH-synthase reaction on substrate (arachidonic acid, O2 and homovanillic acid) concentrations was studied, and the corresponding Km values were determined. The effect of Tween-20 and Lubrol PX concentrations on the reaction rate were examined. It was shown that with a decrease in the surfactant concentration the reaction rate increases.     

Effects of fluid shear on immobilized enzyme kinetics.

There have been a number of reports concerning the damaging effects of shear on globular proteins in solution. Some recent work has indicated, however, that globular proteins in solution are relatively stable, but may be inactivated at air-liquid interfaces during shearing. This study investigated the effects of fluid shear on immobilized enzyme activity. Immobilized enzyme reactors were built to operate with the enzyme immobilized at the boundary of a fluid flow field. Two different enzymes, penicillinase and lactate dehydrogenase, were covalently bound to the interior surface of nylon tubes. Fluid shear rate was changed by varying the flow rate of substrate (reactant) solution through the tube, and fluid shear stresses were increased by increasing the viscosity of the recirculating solution. There were no observed effects of fluid shear on immobilized penicillinase or lactate dehydrogenase activity at shear rates of up to 10,350 s-1 or at shear stresses of up to 73 Pa.     

The origins of enzyme kinetics

The equation commonly called the Michaelis–Menten equation is sometimes attributed to other authors. However, although Victor Henri had derived the equation from the correct mechanism, and Adrian Brown before him had proposed the idea of enzyme saturation, it was Leonor Michaelis and Maud Menten who showed that this mechanism could also be deduced on the basis of an experimental approach that paid proper attention to pH and spontaneous changes in the product after formation in the enzyme-catalysed reaction. By using initial rates of reaction they avoided the complications due to substrate depletion, product accumulation and progressive inactivation of the enzyme that had made attempts to analyse complete time courses very difficult. Their methodology has remained the standard approach to steady-state enzyme kinetics ever since.     

The kinetics of enzyme inactivation

The course of hydrolysis of β-glycerophosphate catalyzed by a group of different enzyme extracts, both with and without the addition of Mg, with and without preincubation of the enzyme, has been studied and the results discussed on the basis of a mathematical analysis. In all the extracts, it appears that two distinct and independently acting constituent enzymes—or perhaps “principles” of the same enzyme—are present, one acting much more rapidly but also more rapidly inactivated than the other. Storage in the refrigerator changes markedly the behavior of both constituents, though in different ways. There is evidence that in some cases an enzyme is limited in its hydrolytic “capacity” in the sense that after an enzyme molecule has decomposed a definite number of substrate molecules, it thereafter becomes entirely passive. Further, there is evidence, in the case of one extract, that the roles of catalytically more and less active constituents in the absence of Mg are reversed in its presence. Finally, a damped periodicity is found which indicates the presence of two factors of an unknown sort which influence and are influenced by the inactivation of the enzyme.