Substrate binding properties of L-glycerol-3-phosphate dehydrogenase in isolated liver mitochondria of hyperthyroid rats.

In isolated, intact liver mitochondria from hyperthyroid rats, the L-glycerol-3-phosphate binding site(s) of the L-glycerol-3-phosphate dehydrogenase was (were) found to be influenced by the nature of the electron acceptor, as well as by the pH and the presence of calcium ions. With the hydrophobic electron acceptor menadione a single L-glycerol-3-phosphate binding site was detected kinetically at bulk pH values between 6.5 and 9.0. With the hydrophilic phenazine methosulfate, on the other hand, two L-glycerol-3-phosphate binding sites were distinguishable at pH > or = 7.5 and pH > or = 7.0, in the presence and absence of Ca2+, respectively. The kinetic mechanism of the reaction catalyzed by L-glycerol-3-phosphate dehydrogenase is ping pong Bi Bi with a hydrophilic electron acceptor, where as with the hydrophobic substance, a sequential Bi Bi mechanism was observed. We suggest that the latter mechanism has physiological relevance.

Coupled enzymatic reactions measured in a single protein crystal from myogen A.

Pairwise coupled reactions of fructose-1,6-bisphosphate aldolase and sn-glycerol-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and D-glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and sn-glycerol-3-phosphate dehydrogenase have been detected by microspectrophotometry in single crystals obtained from myogen A in the presence of polyethylene glycol. Microspectrophotometric measurements with polarized light demonstrate that the protein molecules are oriented and that NADH is bound with a definite orientation to the dehydrogenases within the crystal.

Double inhibition of L-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 other's 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.

Transient-time analysis of substrate-channelling in interacting enzyme systems.

The kinetics of dynamically interacting enzyme systems is examined, in the light of increasing evidence attesting to the widespread occurrence of this mode of organization in vivo. The transient time, a key phenomenological parameter for the coupled reaction, is expressed as a function of the lifetime of the intermediate substrate. The relationships between the transient time and the pseudo-first-order rate constants for the coupled reaction by the complexed and uncomplexed enzyme species are indicative of the mechanism of intermediate transfer ('channelling'). In a dynamically interacting enzyme system these kinetic parameters are composite functions of those for the processes catalysed by the complex and by the separated enzymes. The mathematical paradigm can be extended to a linear sequence of N coupled reactions catalysed by dynamically (pair-wise) interacting enzymes.

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.

Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium.

Ammonium sulfate, a typical component of crystallization media of proteins, stabilizes an inactive conformation of pig muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, in the presence of ammonium sulfate the reconstitution of the catalytically active holoenzyme from the apoenzyme and NAD is not instantaneous, as in the case of enzymes from Bacillus stearothermophilus and the Mediterranean lobster Palinurus vulgaris. With pig muscle enzyme, at pH 6.0, the time course of formation of the characteristic Racker band can be monitored by a rapid mixing stopped flow technique. Activation follows a single exponential curve with a rate constant independent of the concentration of both NAD and protein and, therefore, appears to be limited by a slow protein isomerization (k = 7 +/- 2 s-1). Accordingly, when the apoenzyme is simultaneously exposed to NAD and either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate, the ensuing reactions (the redox and the acylation steps, respectively) are kinetically limited by the same protein isomerization. At pH 7.0 and 8.0, however, two among the four active sites react with NAD at an unmeasurably high rate, while the other two sites behave as they do at pH 6.0. When the pig muscle apoenzyme is preincubated and allowed to react with either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate before the rapid mixing with NAD, both the redox reaction and the NAD-dependent activation of apo-acyl-enzyme toward arsenolysis become unmeasurably fast. Similarly, when the sulfate in the medium is replaced by ions such as phosphate and citrate, the reconstitution of the active holoenzyme is practically instantaneous. Thus, the slow protein isomerization observed in the presence of sulfate and abolished by competing substrates and anions is diagnostic of a structural state of the pig muscle apoenzyme, which is induced by sulfate ions bound within the enzyme active site.

The control of cell metabolism for homogeneous vs. heterogeneous enzyme systems.

Metabolic control theories, based on such parameters as "elasticity coefficients" and "flux-control coefficients", have emerged in recent years. These offer a potentially unifying, holistic paradigm for understanding the regulation of cell metabolism. Much of the foundation relies on the supposition that the system is a homogeneous bulk-phase solution of individual enzymes. We examine some of the tenets of such theories, in the light of increasing knowledge of enzyme organization in vivo. We cast the control parameters into a more general form applicable to the linear kinetic regime, using a newly defined unit--the kinetic power, which allows complete specification in terms of any and all factors which bear upon the conversion of free substrate to free product in situ. Extending the control theory to heterogeneous states of enzyme organization, we make a formal distinction between "solution connectivity" and "structural connectivity" in a multienzyme system. The use of "structural" rate expressions leads to the definition of a flux-control coefficient which specifies the interdependence of the individual rate processes in an organized system. The problems and limitations in applying the control theory to experimental analysis of real systems in situ are discussed. "We have arrived at last at a point which comes rather close to what might be defined as 'molecular control of cellular activity', only to discover that the 'controlling' molecules have themselves acquired their specific configurations, which are the key to their power of control, by virtue of their membership in the population of an organized cell, hence under 'cellular control'." (Weiss, 1963).

A kinetic method for distinguishing whether an enzyme has one or two active sites for two different substrates. Rat liver L-threonine dehydratase has a single active site for threonine and serine.

We have elaborated a kinetic method which allows us to evaluate whether a Michaelis-Menten-type enzyme acting on two different substrates has one or two active sites. This method has been used with the rat liver L-threonine dehydratase, which catalyzes the dehydrative deamination of both serine and threonine. The experimental data can be fitted to the theoretical plot obtained for the case of a single active site.

Microenvironment of the enzyme-bound NADH is different in lobster and pig muscle glyceraldehyde-3-phosphate dehydrogenase microcrystals.

Two possible consequences of crystal lattice formation were studied with glyceraldehyde-3-phosphate dehydrogenases isolated from lobster (Palinurus vulgaris) and pig muscle: changes in the microenvironment of the NADH-binding site as detected by fluorescence polarization, and differences in the maximal activities of the microcrystalline enzymes as compared to those in solution. In solution practically no difference was found between the polarization values of the enzyme-NADH and the catalytic intermediate 3-phosphoglyceroyl-enzyme-NADH complexes whether with lobster or with pig enzyme. In microcrystalline state a similar effect was found with the lobster enzyme. However, fluorescence polarization of NADH bound to the pig enzyme was significantly different in the presence and in the absence of the 3-phosphoglyceroyl group. This indicates some change in the microenvironment of the pig enzyme-bound NADH which occurs upon decomposition of the catalytic intermediate. The difference between the microcrystalline lobster and pig muscle glyceraldehyde-3-phosphate dehydrogenases pertains also to their functional properties. Packing of soluble pig muscle enzyme into a crystal lattice stabilizes a unique protein conformation of extremely low activity (about 3% of that measured in solution). The maximal molar activity of the lobster enzyme is identical in crystalline state and in solution, which is an exceptional phenomenon.

On the NADPH dependent reaction of cytoplasmic sn-glycerol-3-phosphate dehydrogenase.

Cytoplasmic sn-glycerol-3-phosphate dehydrogenase (EC. 1.1.1.8.) can reduce dihydroxy acetone phosphate with NADPH as coenzyme under in vitro conditions, in solutions of low ionic strengths, at pH values lower than 7. The reaction is inhibited by phosphoenolpyruvate, NAD, ATP, ADP and Pi. In the cell this reaction can occur apparently only in case of specific metabolic conditions, i.e. when the local pH is low and the enzyme is protected from the inhibition by the above listed metabolites.

Two rules of enzyme kinetics for reversible Michaelis-Menten mechanisms.

In a Michaelis-Menten type reversible enzyme reaction (one substrate, one product) the rapid equilibrium kinetics in one direction excludes rapid equilibrium in the reverse direction. If rapid equilibrium functions in any direction, in the reverse reaction van Slyke type 'kinetic constant' appears in the rate equation independently of whether steady state is reached in finite time or the final equilibrium is attained at t = infinity. If the reaction proceeds in one direction with rapid equilibrium and in the reverse direction with steady-state kinetics, the thermodynamic equilibrium of the reaction determines that a higher equilibrium concentration of product (or substrate) can be reached only with steady-state kinetics.

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.

Stability, heat stability and heat sensitivity of proteins: thermodynamic considerations.

The heat sensitivity and stability of a protein should be characterized by the Arrhenius parameters of the irreversible denaturation. The terms "thermostability" or "activation enthalpy" and "activation entropy" of denaturation as "thermodynamic parameters of protein stability" are misleading. The terms "measure of heat sensitivity" and "measure of stability" should be used as comparative data for protein structure.

The evolution of enzyme kinetic power.

Evolution of the kinetic potential of enzyme reactions is discussed. Quantitative assessment of the evolution of enzyme action has usually focused on optimization of the parametric ratio kcat./Km, which is the apparent second-order rate constant for the reaction of free substrate with free enzyme to give product. We propose that the general form kcat.[E]T/Km (where [E]T is total enzyme concentration), which is designated the 'kinetic power', is the real measure of kinetic/catalytic potential in situ. The standard paradigm of 'perfection' dictates the evolutionary maximum of 'kinetic power' to be k+s[E]T/2, where k+s is the diffusion-controlled rate constant for formation of the ES complex (and, hence, for the overall enzyme reaction). We discuss the role of protein conformational mobility in determining this state of 'perfection', via gating of substrate binding and determination of the catalytic configuration. Going beyond the level of the individual enzyme, we indicate the manner by which the organizational features of enzyme action in vivo may enhance the 'kinetic power'. Through evolutionary 'perfection' of the microenvironment, one finds that the 'kinetic power' of enzymes can be affected by alteration of [E]T as well as the unitary rate constants. At this level of complexity, we begin to realize that the 'kinetic' description of cell metabolism must be supplemented with thermodynamic concepts.

Errors in the evaluation of Arrhenius and van't Hoff plots.

Errors in the numerical values of activation or normal enthalpies, entropies and free enthalpies calculated from Arrhenius or van't Hoff plots, respectively, are due to the neglect of equidimensionality in equations, or to inappropriate approximations. The logarithmization of dimensioned quantities should be avoided, which demands the use of relative concentrations if a change in mole number occurs in the reaction. The application of the Arrhenius plot to enzymic reactions by using Vmax./ET instead of the rate constant of product formation has meaning only if the reaction follows the simplest Michaelis-Menten mechanism; however, the use of the van't Hoff plot using Km is questionable even in the latter case.

Homologous partial sequences in dehydrogenases.

Such details of the primary structure were sought that are common in all dehydrogenases of known amino acid sequence. Twenty-six sequences of eight kinds of dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase, glycerol-3-phosphate dehydrogenase, ribitol dehydrogenase, L-hydroxyacyl-CoA dehydrogenase and homoserine dehydrogenase) have been compared by the aid of the artificial intelligence language Prolog, the amino acids being classified into groups according to their chemical properties, and alpha-helix or beta-sheet-forming abilities. We found tetrapeptides that occurred in all dehydrogenases examined. By using these tetrapeptides as markers a population of 84 partial sequences has been described. The partial sequences constituting this population are peptides comprising 35 residues. It has been shown statistically that these peptides form a homogeneous sample as regards the frequency of occurrence of amino acid groups. This statistically homogeneous partial sequences can be regarded as homologous and it is assumed that their presence is characteristic of dehydrogenases.