Tamás Keleti

On the “cytosociology” of enzyme action in vivo: A novel thermodynamic correlate of biological evolution

Abstract The evolution of enzyme action in vivo is examined, in the light of established thermodynamic correlates of biological evolution. Adopting a “process” view of matter in the “living state,” the authors focus the analysis on the transition-state theory of reaction rates. Thus, the free-energy change associated with the transition-state barrier is seen as a primary target in the evolution of cellular metabolism. The utility and limitations of reductionistic approaches to enzyme evolution, based on the single enzyme, are explored first. Then, canvassing the wealth of evidence on the role of enzyme organization in vivo , the authors synthesize a “cytosociological” view of enzyme evolution. In this view, a composite (resultant) of individual transition-state barriers is deemed a more appropriate “potential function” for modification in the higher evolution of cell metabolism. The suggested direction of evolutionary changes in this function, dictated by the increasing “socialization” of enzyme action in vivo , stands as a novel postulate. This approach is shown to be completely consonant with current thinking on the thermodynamics of biological evolution, and to provide further insight into the nature of material transformations in the “living state”.

Macromolecular interactions in enzyme regulation

Abstract The differences observed by several authors in the NaDH-binding ability of the subunits of glyceraldehyde-3-phosphate dehydrogenase are caused by the dissociation of the enzyme. A specific model has been elaborated for the interpretation of the phenomenon; the earlier interpretation in terms of ligand-induced negative cooperatively seems to be erroneous. In the regulation of the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase in the glycolytic pathway the association-dissociation dynamics of the enzyme play the decisive role. In the higher aggregational forms of the enzyme the relative concentration of “functioning active centres” progressively decrease and this may furnish the basis for a novel type of regulatory mechanism. Aldolase forms a complex with glyceraldehyde-3-phosphate dehydrogenase (presumably with the dimeric form) thereby enhancing the activity of the latter. This finding calls the attention to the importance of enzyme-enzyme interactions in the intracellular metabolic regulation. The modelling of enzyme-other macromolecule interactions was performed by studying the behavior of aldolase and glyceraldehyde-3-phosphate dehydrogenase in the solution of synthetic polymers. The change of kinetic parameters suggests that the interaction induces conformational alterations in the enzymes. The kinetic consequences of structural changes depend not only on the enzyme, but also on the structure of the polymer.

Effect of association-dissociation on the catalytic properties of glyceraldehyde 3-phosphate dehydrogenase

Abstract The enzymatic activity of d -glyceraldehyde 3-phosphate dehydrogenase depends nonlinearly on protein concentration in the range 3 × 10 −8 to 3 × 10 −6 m . With increasing enzyme concentrations the apparently hyperbolic substrate saturation curves turn into sigmoidal ones. From the kinetic and physicochemical data it is assumed that the enzyme exists as an equilibrium mixture of different oligomeric states. The system is found to be consistent with a model characterized by rapid equilibrium between monomer-dimer-tetramer, the tetramer being inactive, assuming identical intrinsic binding constants for the substrate in the monomer and in the dimer.

The perfection of substrate-channelling in interacting enzyme systems: energetics and evolution

Some implications of substrate channelling in interacting enzyme systems are considered, with regard to the energetics and evolution of enzyme action. The transient time, a key analytical parameter relating to the phenomenon of channelling, is the basis of our kinetic study. Bounds on the kinetics of multienzyme complexes are established using (apparent) rate constants emanating from the transient-time formulation of coupled reactions. From a transition state representation of the rate process, it is shown how dynamically and statically organized enzyme systems lead to the modification of current ideas on the evolutionary optimization of the energy profile of enzyme catalysis in situ.

Double inhibition ofl-threonine dehydratase by aminothiols

The inhibition of highly purified rat liver L-threonine dehydratase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) by aminothiols (L-cysteine, D-cysteine, cysteamine) has been studied. Single inhibition experiments evaluated by Lineweaver-Burk and Dixon plots showed, in a given concentration range, partially (parabolic) competitive inhibitions, indicating two binding sites for each inhibitor. Double inhibition experiments revealed that the inhibition was antagonistic, the two inhibitors weakening each others effect. Formation of EI1 and EI2 binary complexes, and ESI1, ESI2 and EI1I2 ternary complexes was demonstrated, while formation of the quaternary complex ESI1I2 was ruled out. It is assumed that one inhibitor-binding site coincides with the substrate-binding center while the second inhibitor-binding (allosteric, regulatory) site may comprise the pyridoxal-phosphate-binding SH group(s). The comparison between Km and Ki values and the evaluation of intracellular concentrations of L-threonine, L-cysteine and cysteamine suggest a possible physiological role of the inhibition.

SH groups masked by subunit interaction in glyceraldehyde-3-phosphate dehydrogenase

Abstract The changes in reactivity of the less reactive SH groups of glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) apoenzyme towards thiol reagents were studied during the course of dissociation into dimers and inactivation of the enzyme induced by stoichiometric amounts of ATP. Neither the reactive SH group of Cys-149 nor that of Cys-153 (the latter being the second cysteinyl group in order of reactivity towards thiol reagents) is ozidized during dissociation and inactivation caused by ATP treatment. However, during this process one SH group per subunit which was previously masked in the native tetramer is oxidized. This cysteinyl residue corresponds to Cys-281 as identified anallytically. The molecular weight of the ATP-treated enzyme is 60 000 even in the presence of 6 M urea. Both enzymatic activity and the oxidized SH group can be fully recovered by treatment with 2-mercaptoethanol. We conclude that during dissociation into dimers by ATP, residues Cys-281 form interchain S-S bridges within the dimers.

Determination of Michaelis-Menten parameters from initial velocity measurements using unstable substrate

By initial velocity measurements and two different methods of plotting the experimental data, the Km and Vmax of enzyme action and the first-order rate constant of substrate decomposition can be determined simultaneously under the same conditions. This method permits the determination of Km and Vmax even if the presence of the enzyme (or any impurity in the solutions used) influences the rate of substrate decomposition. The theoretical treatment was proved by determining the Michaelis-Menten parameters of D-glyceraldehyde-3-phosphate dehydrogenase and the first-order rate constant of hydrolysis of the unstable substrate, bisphosphoglycerate.

Coupled Reactions and Channelling: their Role in the Control of Metabolism

Control analysis was developed for describing the regulatory properties of metabolic pathways (Savageau, 1972, 1976; Kacser & Burns, 1973, 1979; Kacser, 1983; Heinrich & Rapoport, 1973, 1974ao, 1983; Heinrich et al, 1977). The more effective the control, the more elastic the metabolic pathway, i.e more able to respond to changes in external conditions. Kacser (1983) has suggested the idea of “molecular democracy” to characterize each enzyme in a metabolic process as an autonomous entity and the control as a sort of linear superposition of the effects of the individual enzymes. The milieu of this “molecular society” is a bulk aqueous solution with non-interacting enzymes and non-compartmentalized metabolites homogeneously dispersed therein. The links in such a metabolic network are the intermediate metabolite pools.

Kinetic power: Basis of enzyme efficiency, specificity and evolution

Abstract The kinetic power of an enzyme reaction is defined as the ratio of maximal velocity to the Michaelis constant. The maximal velocity and the kinetic power are the independent parameters of catalytic action. The kinetic power allows complete specification in terms of any and all factors which bear upon the conversion of free substrate to free product in situ. The transient time of coupled reactions is the reciprocal of the kinetic power. The transient time may be expressed in terms of the lifetime of the intermediate in the case of both non-interacting and interacting enzyme systems, whether the interaction is static or dynamic. The formation of enzyme complexes may lead to channelling, and consequently to a decrease in the transient time. Some implications of substrate channelling in interacting enzyme systems are considered with regard to the energetics and evolution of enzyme action.

Enzyme immobilization by poly(vinyl alcohol) gel entrapment

Abstract A method is presented for gel entrapment of enzymes [1], consisting of the formation of poly(vinyl alcohol) film from enzyme-containing poly(vinyl alcohol) solution by drying or salt treatment. The yield of immobilization can be enhanced by adding materials protecting the enzymes (inert proteins, —SH— compounds, etc.) to the gel-forming solution to decrease enzyme leakage. The details of immobilization procedure and some properties of aldolase and neutral lactase immobilized in this manner are described.