József Batke

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.

How to determine the efficiency of intermediate transfer in an interacting enzyme system

A kinetic method, based upon measuring the transient time of coupled reactions, is proposed for the determination of the intermediate channel efficiency in a system of functionally interacting enzymes. The procedure rests upon a novel description in which the transient time is expressed as a function of channel efficiency and lifetime of the intermediate molecules. By this approach the reduction of transient time can be explained even if no changes in the kinetic parameters of the individual reactions occur. For determining channel efficiency, a linearized form has been evaluated and applied to the analysis of the kinetics of the aspartate aminotransferase‐glutamate dehydrogenase coupled reaction, for which the data were taken from the literature [(1982) Eur. J. Biochem. 121, 511–517].

Remarks on the supramolecular organization of the glycolytic system in vivo.

The great latent catalytic capacity, manifested at the extremely high intracellular concentrations and in large apparent k cat/K m values, of the glycolytic enzymes on the one hand and their tendency in experiments in vitro to form functionally‐specific flux‐enhancing (channeling) complexes on the other, is considered and discussed as an apparent discrepancy. A random association of glycolytic enzymes in vivo is probable.

Complex of brain d-phosphoglycerate mutase and γ enolase and its reactivation by d-glycerate 2,3-bisphosphate

The dissociabilities of dimeric gamma enolase, alpha enolase, and phosphoglycerate mutase of brain origin were tested using fluorescein isothiocyanate attached covalently to these enzymes. The dissociation constant of dimeric gamma enolase is lower (Kd = 0.03 microM) than that of the alpha enolase (Kd = 3 microM), while dimeric mutase seems to be nondissociable in the concentration range 0.1-10 microM, at pH 7.3 in 50 mM imidazole buffer at 20 degrees C. Interaction of neuron-specific gamma enolase with D-phosphoglycerate mutase was detected with the same fluorescence-labeling technique as well as by a kinetic analysis. The determined dissociation constant of the enolase-mutase complex was found to be in the range 5-40 microM, independent of the technique used. A mixed type of inhibition in the binding of D-glycerate-2-P and mutase to the D-glycerate-2-P binding site on enolase was observed in the absence of D-glycerate-2,3-P2. However, the inhibition of the enolase activity by brain D-phosphoglycerate mutase in the D-glycerate-2-P----phosphoenolpyruvate transformation is almost fully reverted by D-glycerate-2,3-P2, probably via the proper coordination of the active centers in the ternary complex of enolase, D-phosphoglycerate mutase, and their common intermediate, D-glycerate-2-P. The mechanism of intermediate transfer by consecutive enzyme pairs in a nondivergent metabolite flux (around the transformation of D-glycerate-2-P) is examined and conclusions of the present experiments are compared with the results of an extended analysis performed earlier with a divergent metabolite flux (around the transformation of multiusage triosephosphates, D-glyceraldehyde-3-P, and dihydroxyacetone phosphate).

Evidence for absence of an interaction between purified 3-phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase

The possibility of a functional complex formation between glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and 3-phosphoglycerate kinase (EC. 2.7.2.3), enzymes catalysing two consecutive reactions in glycolysis has been investigated. Kinetic analysis of the coupled enzymatic reaction did not reveal any kinetic sign of the assumed interaction up to 4 X 10(-6) M kinase and 10(-4) M dehydrogenase. Fluorescence anisotrophy of 10(-7) M or 2 X 10(-5) M glyceraldehyde-3-phosphate dehydrogenase labeled with fluorescein isothiocynate did not change in the presence of non-labeled 3-phosphoglycerate kinase (up to 4 X 10(-5) M). The frontal gel chromatographic analysis of a mixture of the two enzymes (10(-4) M dehydrogenase) could not reveal any molecular species with the kinase activity having a molecular weight higher than that of 3-phosphoglycerate kinase. Both types of physicochemical measurements were also performed in the presence of substrates of the kinase and gave the same results. The data seem to invalidate the hypothesis that there is a complex between purified pig muscle glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase.

Effect of the concentration of D-glyceraldehyde-3-phosphate dehydrogenase on the reducibility of its firmly bound NAD.

D-glyceraldehyde-3-phosphate dehydrogenase’ (GAPD) is composed of 4 identical subunits [ 1,2] and it binds and also crystallizes with 3.5 to 4 moles NAD per mole of enzyme [3-61. Several authors have found that only two moles of firmly bound NAD per mole of GAPD were reducible [7-lo] or even onlyonemole [ll]. Our present results show that the amount of reducible firmly-bound NAD depends on the concentration of GAPD, when measured with D-glyceraldehyde3-phosphate in the presence of arsenate ions. Four times recrystallized GAPD isolated from swine muscle according to Elijdi and Sziirenyi [ 131 was dissolved in 0.1 M glycine buffer pH 8.5. The concentration of the protein was determined by measuring the optical density at 280 nm. The amount of firmly bound NAD was 3.6 mole per mole of GAPD as determined from the change in absorbancy of the GAPDNAD complex at 360 nm (Racker-band) after adding NAD to the GAPD solution (1Oj M). The molecular weight was taken as 140,000 [ 141 . The molar extinction coefficient of NADH at 340 mn is 6.22 X 103 [ 151. D-glyceraldehyde-a-phosphate was prepared from fructose-l ,6diphosphate [ 161 and the contaminating inorganic phosphate was removed as described earlier [ 171. In the assay medium D-glyceraldehyde-3-phosphate was used in 10m3 M concentration in the presence of 10” M arsenate ion. The measured values were corrected for the decrease in absorbancy due to the disappearance of the Rackerband that, in turn, was caused by the transformation of NAD in the GAPD-NAD charge-transfer complex into NADH *. In the fluorimetric determination of NADH another correction was made for the quenching effect of GAPD. The latter was determined inde-

Interaction of rabbit muscle enolase and 3-phosphoglycerate mutase studied by ELISA and by batch gel filtration.

The interaction of rabbit skeletal muscle enolase and 3-phosphoglycerate mutase was detected by an ELISA test, a batch gel-filtration technique, and fluorescence anisotropy measurements, and the activity of enolase was determined to be a function of mutase concentration. The apparent dissociation constant of this enzyme complex is approximately 1 microM. This value seems to be independent of the presence (in fluorescence anisotropy measurements) or the absence (in activity as well as in ELISA experiments) of fluorescein isothiocyanate used widely as a label for determining the complex formation between enzymes in fluorescence anisotropy measurements.

Detailed analysis of heterogeneity of [3H]naloxone binding sites in rat brain synaptosomes

The experiments reported in this paper address the question of heterogeneity of [3H]naloxone binding sites in rat brainstem synaptosomal preparations at 23°C in the presence of 100 mM sodium chloride. Kinetic analysis in the presence of 0.4, 4 and 10 nM [3H]naloxone gave pseudo-first order association rate of 0.9±0.04, 1.23±0.08 and 1.06±0.08 min−1, respectively. The dissociation of a 1 nM [3H]naloxone receptor complex was biphasic with dissociation rate constants of 1.8 and 0.4 min−1. On the other hand, dissociation of 10 nM [3H]naloxone was monophasic with akd of 1.1 min−1. Two subpopulations of binding sites were also observed by steady state binding studies, with Kd values of 0.5 and 3.4 nM and a ratio of high to low affinity sites of 1:9. Heterogeneity of [3H]naloxone binding sites could be seen by displacement studies performed with opioid eptides and alkaloids. We suggest that our data best fits a model with two independent naloxone binding sites wherein either one or both undergo a multi-step interaction with ligand.

Modelling of allosteric interactions in dissociable tetrameric isoenzyme systems

Abstract Allosteric (intersubunit) interactions were analysed by using a simple model for the case of dissociable tetrameric isoenzyme systems built up of two types of subunit. The model permits calculation of the interaction constants under certain conditions, whereas in other cases only their ratios can be determined. By this approach one can decide which form(s) of the dissociable oligomer is(are) enzymically active, independent of the types of interaction in the tetrameric molecule.