Juan J. Aragón

Regulation of enzyme activity in the cell: effect of enzyme concentration.

The rapid development in our understanding of the regulation of enzyme activity makes it a high priority to ascertain whether the behavior of purified enzymes reflects their functional characteristics in vivo. Enzyme concentration is usually the most significant difference between routine in vitro assays and in vivo conditions, as it is well known that many intracellular enzymes are present in vivo at much higher concentrations than used in vitro. Various procedures are suitable for kinetic analysis at physiological concentrations of enzyme. Those more frequently used have been cell per‐meabilization, the utilization of purified enzymes at concentrations close to the in vivo range, and the addition of polyethylene glycol to increase the local protein concentration. In this review we briefly summarize observations on enzymes reported to exhibit concentration‐dependent activity. The effect of enzyme concentration has been most thoroughly investigated in the case of phosphofructokinase. These studies may provide insight into the regulation of this important enzyme in the cell. The implications of both homologous and heterologous protein‐protein interactions for the effect of enzyme concentration and their roles in the control of enzyme activity in vivo are also discussed.—Aragon, J. J.; Sols, A. Regulation of enzyme activity in the cell: effect of enzyme concentration. FASEB J. 5: 2945‐2950; 1991.

Analysis of phosphofructokinase subunits and isozymes in ascites tumor cells and its original tissue, murine mammary gland

Phosphofructokinase (PFK) subunits and isozymes have been examined in ascites tumor cells and murine mammary gland, the tissue from where this tumor originated. Ascites tumor was found to contain predominantly the C‐type subunit, whereas the L‐type subunit was more abundant in mammary gland. An altered M‐type subunit of lower electrophoretic mobility was found in both cell types and no M4 homotetramers were detected in either of them. Characteristic regulatory properties of ascites tumor PFK, i.e. insensitivity to fructose‐1,6‐P2 activation and inhibition by P‐enolpyruvate, were also observed in the mammary gland isozyme. The nature of these properties and the contribution of the distinct subunit types to fructose‐1,6‐P2 activation are discussed.

4-O-β-spD-Galactopyranosyl-spD-xylose: A new synthesis and application to the evaluation of intestinal lactase

4-O-beta-D-Galactopyranosyl-D-xylose (2) was prepared from benzyl 2,3-O-isopropylidene-beta-D-xylopyranoside by glycosylation with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl bromide and subsequent deprotection. Compound 2 was hydrolyzed in vitro by intestinal lactase; the Vmax was 25% of that with lactose and the Km was 370mM (cf. 27mM for lactose). Oral administration of 2 suckling rats led to urinary excretion of D-xylose which could be estimated colorimetrically.

Mitochondrial Transcription Factor B2 Is Essential for Metabolic Function in Drosophila melanogaster Development

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.

Specific activation by fructose 2,6-bisphosphate and inhibition by P-enolpyruvate of ascites tumor phosphofructokinase

Abstract Phosphofructokinase of Ehrlich ascites tumor is very sensitive to activation by fructose 2,6-bisphosphate while it is unaffected by fructose 1,6-bisphosphate. This finding shows that in this enzyme a specific site for fructose 2,6-bisphosphate exists and suggests that the isozymes sensitive to both bisphosphates are likely to have two different regulatory sites for these compounds. The tumor isozyme has also been found to be allosterically inhibited by P- enol pyruvate in contrast with the muscle enzyme.

Phosphofructokinase and fructosebisphosphatase from muscle can interact at physiological concentrations with mutual effects on their kinetic behavior

Phosphofructokinase (PFK) and fructosebisphosphatase (FBPase) from muscle studied at physiological concentrations have been found to influence the kinetic behavior of each other. Under these conditions PFK can be activated up to ca. 4-fold by FBPase, while the latter can be inhibited up to ca. 3-fold by the former. Diluted enzymes did not interact with each other; nevertheless, they did so in the presence of polyethylene glycol. Equimolar amounts of either glucosephosphate isomerase or aldolase had no effect on concentrated PFK. The kinetic interactions between PFK and FBPase should be taken into account for fuller understanding of their regulatory behavior in vivo.

Functional complementation of yeast phosphofructokinase mutants by the non-allosteric enzyme from Dictyostelium discoideum

Phosphofructokinase (PFK) from yeast has been replaced by the non‐allosteric isozyme from the slime mold Dictyostelium discoideum. This has been achieved by overexpression of the latter in a PFK‐deficient strain of Saccharomyces cerevisiae under the control of the PFK2 promoter. Transformants complemented the glucose‐negative growth phenotype exhibiting generation times on glucose‐containing media similar to those of an untransformed strain being wild‐type for yeast PFK genes. The PFK produced reacted with an antibody against D. discoideum PFK. It exhibited the same subunit size, quaternary structure and kinetic parameters than those of the wild‐type enzyme, and was also devoid of specific regulatory properties.

Subunit interactions and composition of the fructose 6-phosphate catalytic site and the fructose 2,6-bisphosphate allosteric site of mammalian phosphofructokinase.

Mammalian phosphofructokinase originated by duplication, fusion, and divergence of a primitive prokaryotic gene, with the duplicated fructose 6-phosphate catalytic site in the C-terminal half becoming an allosteric site for the activator fructose 2,6-bisphosphate. It has been suggested that both sites are shared across the interface between subunits aligned in an antiparallel orientation, the N-terminal half of one subunit facing the C-terminal half of the other. The composition of these binding sites and the way in which subunits interact to form the dimer within the tetrameric enzyme have been reexamined by systematic point mutations to alanine of key amino acid residues of human muscle phosphofructokinase. We found that residues His-199, His-298, Arg-201, and Arg-292 contribute to the catalytic site and not to the allosteric site, because their mutation decreased the affinity for fructose 6-phosphate without affecting the activation by fructose 2,6-bisphosphate or its binding affinity. In contrast, residues Arg-566, Arg-655, and His-661 were critical components of the fructose bisphosphate allosteric site, because their mutation strongly reduced the action and affinity of the activator, with no alteration of substrate binding to the active site. Our results suggest that mammalian phosphofructokinase subunits associate with the N-terminal halves facing each other to form the two catalytic sites/dimer and the C-terminal halves forming the allosteric sites. Additionally, mutation of certain residues eliminated activation by fructose 1,6-bisphosphate, but not its binding, with little effect on activation by fructose 2,6-bisphosphate, indicating a divergence in the signal transduction route despite their binding to the same site.

Effect of polyethylene glycol on the kinetic behaviour of pyruvate kinase and other potentially regulatory liver enzymes

Assay in the presence of 10% polyethylene glycol has been systematically used with potentially regulatory liver enzymes as an indirect way to induce aggregation of enzymes corresponding to that which could occur at their physiological concentrations. Pyruvate kinase L was markedly affected by polyethylene glycol, as was muscle phosphorylase a, while pyruvate kinase M as well as glucokinase, fructose‐1,6‐bisphosphatase and other liver enzymes examined were not affected.

A direct enzymatic synthesis of β-d-galactopyranosyl-d-xylopyranosides and their use to evaluate rat intestinal lactase activity in vivo

By enzymatic beta-D-galactosylation of D-xylose a mixture of 4-, 3-, and 2-O-beta-D-galactopyranosyl-D-xyloses (1, 4, and 7, respectively) was obtained in 50% isolated yield. Disaccharides 1, 4, and 7 are substrates of intestinal lactase isolated from lamb small intestine with K(m) values of 250.0, 4.5, and 14.0 mM, respectively. The mixture was used to monitor the normal decline in lactase activity in rats that takes place after weaning. The data obtained by this method correlated with the levels of intestinal lactase activity in the same animals after being sacrificed.