Biomedical subjects

P Tompa

Publications and source records attributed to P Tompa.

26 records · Page 2Linked to original sources

PubMed1990

The mechanism of succinate or fumarate transfer in the tricarboxylic acid cycle allows molecular rotation of the intermediate.

Mitochondria were incubated with L[5-13C]glutamic acid and the distribution of the label between the two carboxyl carbon atoms of the L-aspartic acid formed was determined by 13C NMR. The reaction sequence leading from L-glutamic acid to L-aspartic acid spans the tricarboxylic acid cycle reactions involving the two symmetrical intermediates succinate and fumarate. The C2 symmetry of these intermediates in principle permits a discrimination of the mechanism of their transfer between their enzyme sites of production and utilization. A direct transfer of metabolite from site to site by translation alone predicts an unequal distribution of 13C between the C1 and C4 of aspartate, whereas molecular rotation during transfer allows for a scrambling of the original C5 label. Under several conditions of different glutamate concentrations and solvent osmotic pressures, equal labeling in the C1 and C4 carbons of aspartate is observed. This observation is inconsistent with a transfer mechanism restricting molecular rotation for both intermediates but is compatible with both a random diffusion and a direct transfer mechanism provided the latter allows molecular rotation.

Animals↗

PubMed1990

Fructose-1,6-bisphosphate aldolase preferentially associates to glyceraldehyde-3-phosphate dehydrogenase in a mixture of cytosolic proteins as revealed by fluorescence energy transfer measurements.

Fluorescence energy transfer measurements were implemented for demonstrating the specific character of the interaction between aldolase and glyceraldehyde-3-phosphate dehydrogenase. The enzymes, labeled with monobromobimane (donor) and fluorescein isothiocyanate (acceptor), respectively, were mixed into a cytosol preparation and energy transfer between the two fluorophores was observed to develop. This observation reflecting a contact between the two enzymes, suggests that despite the presence of a multitude of potential macromolecular partners glyceraldehyde-3-phosphate dehydrogenase and aldolase are capable of recognizing each other in the cytoplasm. The idea that in vivo associations of metabolically sequential enzymes may be of physiological benefits is consistent with this result.

Animals↗

PubMed1989

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.

Kinetics↗

PubMed1988

On the appearance and role of a spacer group in the protein amino acids.

Of the 20 protein amino acids, 16 have a methylene group at the beta position, and a further three bear a methine group. No aromatic, carboxamido, carboxylic carbon, or hetero atoms are attached directly to the alpha carbon, but they are separated by this methylene or occasionally by a longer n-alkylene spacer group. Therefore, the structure of the protein amino acids should rather be formulated as H2N-CH((CH2)n-R')-COOH instead of the generally accepted H2N-CH(R)-COOH. The appearance of and the role played by the spacer group are discussed in an evolutionary context. It is suggested that the spacer group appeared as a result of prebiotic selection, based on the relative abundance, racemization rate, and suitability for thermal polymerization of the protein amino acids and their homologs with various spacer group lengths. At the biotic level of evolution the requirements for ribosomal polymerization, as well as the abilities of polypeptides to maintain a stable and flexible three-dimensional structure and to bind ligands are considered and are proposed to have been responsible for the possible exclusion of longer spacer groups. It is concluded that the general role of the spacer group is to ensure the uniformity of the constant regions H2N-CH(-)-COOH and the individuality of the R' contact groups by spatially separating them.

Amino Acids↗

PubMed1987

Quantitation of the interaction between citrate synthase and malate dehydrogenase.

Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase. Assuming 1:1 stoichiometry of the enzymes in the complex, an apparent dissociation constant of 10(-6) M was calculated from fluorescence anisotropy measurements. The effect of various metabolites on the interaction was tested. NAD+, oxalacetate, citrate, ATP, and L(-)- or D(+)-malate had no effect on the association of the two enzymes, whereas alpha-ketoglutarate increased and NADH decreased it. The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected. In kinetic experiments, the activation of malate dehydrogenase by citrate synthase was observed. The effect of pyridine nucleotides and alpha-ketoglutarate is discussed in relation to the direction of the metabolic flow of oxalacetate.

Adenosine Triphosphate↗

PubMed1987

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].

Aspartate Aminotransferases↗

PubMed1986

Interaction of enzymes involved in triosephosphate metabolism. Comparison of yeast and rabbit muscle cytoplasmic systems.

The affinity of baker's yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase was tested by using a fluorescence-probe technique with fluorescein isothiocyanate attached covalently to the enzymes. The dissociation constants of the enzyme-enzyme complexes, as well as the rate constants of association and dissociation, were determined. Data were compared with the parameters derived from a mammalian (rabbit muscle) system, known from the literature and determined under the same conditions (pH 7.5 or 8.5 in 0.05 M Tris/HCl buffer at 20 degrees C). The comparison reveals similarities in the supramolecular organization of these cytoplasmic enzymes in phylogenetically distant species. Moreover, the fact that in vitro hybrid complexes are formed of stability comparable to that of non-hybrid complexes indicates that this ancient characteristic is probably conserved during evolution. A possible regulatory mechanism is presented, based on the dynamic competition, with each other, of the enzymes involved in triosephosphate metabolism.

Animals↗

PubMed1986

The phosphate group of 3-phosphoglycerate accounts for conformational changes occurring on binding to 3-phosphoglycerate kinase. Enzyme inhibition and thiol reactivity studies.

Steady-state kinetic study of the inhibition of 3-phosphoglycerate kinase reaction by the substrate analogues D-glycerol 3-phosphate, 2-phosphoglycolate, tartronate and malonate revealed competition with respect to 3-phosphoglycerate. D-Glycerate had no detectable inhibitory effect. The data indicate that (a) the phosphate of 3-phosphoglycerate plays an essential role in the formation of its complex with the enzyme and, taking into account the relatively strong binding of 3-phosphoglycerate, (b) the two charged groups of the substrate might cause a synergic interaction with the protein. The carboxyl-lacking D-glycerol 3-phosphate is a non-competitive inhibitor with respect to MgATP, while all the investigated carboxyl-containing inhibitors compete for MgATP binding. The inhibitory analogues of 3-phosphoglycerate reduce the reactivity of both the two fast-reacting and the five slow-reacting thiol groups of the enzyme molecule. In the case of the fast-reacting thiols the effect is specifically associated with the presence of a ligand's phosphate group. Similarly mainly the phosphate-containing nucleotides and analogues slow down significantly the reaction rate of the fast-reacting thiols, while adenosine is less effective and the competitive inhibitor adenine has no effect at all. MgADP has an especially dramatic effect as compared to MgATP, in line with the known X-ray structural data. The fast-reacting thiols are of particular interest, since their reactivity is possibly controlled by ligand-induced conformational changes. This is shown by the similar ligand protection against alkylation irrespective of the reagent's electrostatic charge (iodoacetamide or iodoacetate) and also by the similar substrate-binding properties of carboxamidomethylated and the unmodified enzyme.

Animals↗