Ka For Mg2 Research Articles

Major Changes in the Kinetic Mechanism of AMP Inhibition and AMP Cooperativity Attend the Mutation of Arg49 in Fructose-1,6-bisphosphatase

The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.

Mutation of arginine 276 to methionine changes Mg2+ cooperativity and the kinetic mechanism of fructose-1,6-bisphosphatase.

Arginine 276 of porcine liver fructose-1,6-bisphosphatase (FBPase) was mutated to methionine by site-directed mutagenesis on the basis of the crystal structure of the enzyme [Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., & Lipscomb, W.N. (1993) Biochemistry 32, 1844-1857]. The mutant and wild-type forms of the enzyme were purified to homogeneity and characterized by circular dichroism spectrometry (CD) and initial-rate kinetics. There were no discernible differences between the secondary structures of the wild-type and the mutant enzymes on the basis of the CD data. Replacement of Arg 276 with methionine caused a significant decrease in the enzyme's activity. The kcat for the mutant enzyme was only about 0.67% of that of the wild-type enzyme. Most importantly, the mutation caused the total loss of cooperativity for Mg2+ and changed the kinetic mechanism to one in which the substrate adds to FBPase before Mg2+ and in which all steps equilibrate rapidly relative to the conversion of the ternary complex of enzyme, substrate, and Mg2+ to products. The Ka for Mg2+ increased by only about 5-fold relative to that of the wild-type enzyme. The mutation did not change the Ki for AMP or the Hill coefficient of this allosteric inhibitor. The Ki for fructose 2,6-bisphosphate was increased by 16-fold compared with that of the wild-type enzyme. The Km for fructose 1,6-bisphosphate was similar to that of the wild-type enzyme. It is concluded that Arg 276 is critical for activity and Mg2+ cooperativity with FBPase and it determines the enzyme's kinetic mechanism.

Glycine 122 Is Essential for Cooperativity and Binding of Mg2+ to Porcine Fructose-1,6-bisphosphatase

Site-directed mutagenesis of an amino acid residue in the substrate binding site of porcine fructose-1,6-bisphosphatase was carried out based on the crystal structure of the enzyme (Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W. N. (1993) Biochemistry 32, 1844-1857). A mutant enzyme form of fructose-1,6-bisphosphatase, G122A, was purified and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectrometry (CD), and initial rate kinetics. There were no discernible differences between the secondary structures of the wild-type and the mutant enzyme on the basis of CD data. Altering Gly-122 to alanine caused a significant decrease in the enzyme's activity and affinity for Mg2+. The kcat for this mutant enzyme was only about 5% of that of wild-type fructose-1,6-bisphosphatase, and the Ka for Mg2+ was about 20-fold higher than that of the wild-type enzyme. The Ki for AMP was increased 77-fold in the case of the mutant enzyme; however, the Hill coefficient was unaltered. Most importantly, it was observed that replacement of Gly-122 with alanine caused the total loss of cooperativity for Mg2+. It is concluded that Gly-122 is essential for Mg2+ cooperativity and important for binding of Mg2+ and AMP as well as for enzyme activity.

Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin.

An expression plasmid containing a hybrid gene encoding a protein having the primary amino acid sequence of the adenylate cyclase from Escherichia coli was constructed. When the gene was induced, the adenylate cyclase could be expressed at high levels in a cya- strain of E. coli. The majority of the enzymatic activity and protein (having a molecular weight of 95,000) induced was insoluble. However, treatment of the insoluble fraction of cell lysates with trypsin resulted in both an increase in and solubilization of the total amount of adenylate cyclase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the soluble protein produced by treatment with trypsin revealed a polypeptide having a molecular weight of 30,000. This soluble, catalytically active fragment of adenylate cyclase was purified and subjected to amino-terminal sequence analyses; two amino-terminal sequences were identified beginning at residue 82 and at residue 342 of the intact enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified fragment followed by either silver or Coomassie Blue staining revealed the presence of only a single polypeptide having a molecular weight of 30,000; a short oligopeptide associated with the amino terminus at residue 342 could not be detected. Site-directed mutagenesis was used to place a stop codon at residue 341; the truncated enzyme was catalytically active, so the short oligopeptide is not necessary for catalysis. The Km for ATP, the Ka for Mg2+, and the Vmax determined for the product containing the 30,000-dalton fragment were similar to the values reported for the intact enzyme from E. coli.

Regulation of magnesium but not calcium transport by phorbol ester.

Phorbol esters in the presence of Ca2+ apparently mimic diacylglycerol in activating protein kinase C. Resulting phosphorylations alter multiple cellular processes including inhibition of the action of Ca2+-mobilizing agonists. In contrast to this inhibition of Ca2+ mobilization, addition of 4 beta-phorbol 12-myristate 13-acetate (PMA) to murine S49 lymphoma cells stimulated Mg2+ influx severalfold without any detectable alteration of Mg2+ efflux or of Ca2+ influx or efflux. Stimulation of Mg2+ influx did not require extracellular Ca2+, was half-maximal at 10 nM PMA, and was characterized by a marked increase in the Vmax of Mg2+ influx without change in the Ka for Mg2+. Stimulation of Mg2+ influx was not mimicked by 4 alpha-phorbol didecanoate, which does not activate protein kinase C and was not the result of Na+/H+ exchange activity. The effect of PMA on Mg2+ influx was inhibited by the beta-adrenergic agonist (-)-isoproterenol, which we have previously shown to inhibit Mg2+ influx by a non-cyclic AMP-dependent mechanism (Maguire, M. E., and Erdos, J. J. (1980) J. Biol. Chem. 255, 1030-1035). Forskolin, a direct activator of adenylate cyclase, also inhibited PMA stimulation of Mg2+ influx, suggesting the presence of both cyclic AMP-dependent and -independent influences on Mg2+ influx. We have also previously demonstrated that Mg2+ influx occurs solely into a small subcytoplasmic pool (Grubbs, R. D., Collins, S. D., and Maguire, M. E. (1984) J. Biol. Chem. 259, 12184-12192). PMA did not alter this compartmentation; rather, it almost doubled the content of this cytosolic Mg2+ pool. These data indicate that, in addition to phorbol ester modulation of intracellular Ca2+ mobilization, substantial changes in Mg2+ flux and content occur. They further demonstrate that Mg2+ influx is regulated by a variety of stimuli. It seems likely that such alterations in Mg2+ flux and content would have physiological consequences.

Des-1-25-fructose-1,6-bisphosphatase, a nonallosteric derivative produced by trypsin treatment of the native protein.

Limited tryptic digestion of pig kidney fructose-1,6-bisphosphatase in the presence of magnesium ions results in the formation of an active enzyme derivative which is no longer inhibited by the allosteric effector AMP. The presence of AMP during incubation of fructose-1,6-bisphosphatase with trypsin protects against the loss of AMP inhibition. By contrast, the presence of the nonhydrolyzable substrate analog fructose 2,6-bisphosphate accelerates the rate of formation of that form of fructose-1,6-bisphosphatase which is insensitive to AMP inhibition. Sodium dodecyl sulfate-polyacrylamide electrophoresis of samples taken during trypsin treatment shows that the loss of AMP inhibition parallels the conversion of the native 36,500 molecular weight fructose-1,6-bisphosphatase subunit into a 34,000 molecular weight species. Automated Edman degradation of trypsin-treated fructose-1,6-bisphosphatase following gel filtration shows a single sequence beginning at Gly-26 in the original enzyme, but no changes in the COOH-terminal region of fructose-1,6-bisphosphatase. Thus, the proteolytic product has been characterized as "des-1-25-fructose-1,6-bisphosphatase." A comparison of the kinetic properties of control enzyme and des-1-25-fructose-1,6-bisphosphatase reveals some differences in properties (pH optimum, Ka for Mg2+, K+ activation, inhibition by fructose 2,6-bisphosphate) between the two enzymes, but none is so striking as the complete loss of AMP sensitivity shown by des-1-25-fructose-1,6-bisphosphatase. The loss of AMP inhibition is due to the loss of AMP-binding capacity, but it is not known at this stage whether residues of the AMP site are present in the 25-amino acid NH2-terminal region or the removal of this region leads to a conformational change that abolishes the function of an AMP site located elsewhere in the molecule.

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.

Autophosphorylation of phosphorylase kinase. Divalent metal cation and nucleotide dependency.

This study reports on the divalent metal ion specificity for phosphorylase kinase autophosphorylation and, in particular, provides a comparison between the efficacy of Mg2+ and Mn2+ in this role. As well as requiring Ca2+ plus divalent metal ion-ATP2- as substrate, both phosphorylase kinase autoactivation and phosphorylase conversion are additionally modulated by divalent cations. However, these reactions are affected differently by different ions. Phosphorylase kinase-catalyzed phosphorylase conversion is maximally enhanced by a 4- to 10-fold lower concentration of Mg2+ than is autocatalysis and, whereas both reactions are stimulated by Mg2+, autophosphorylation is activated by Mn2+, Co2+, and Ni2+ while phosphorylase a formation is inhibited. This difference may be due to an effect of free Mn2+ on phosphorylase rather than the inability of phosphorylase kinase to use MnATP as a substrate when catalyzing phosphorylase conversion since Mn2+, when added at a level which minimally decreases [MgATP], greatly inhibits phosphorylase phosphorylation. The interactions of Mn2+ with phosphorylase kinase are different from those of Mg2+. Not only are the effects of these ions on phosphorylase activation opposite, but they also provoke different patterns of subunit phosphorylation during phosphorylase kinase autocatalysis. With Mn2+, the time lag of phosphorylation of both the alpha and beta subunits of phosphorylase kinase in autocatalysis is diminished in comparison to what is observed with Mg2+, and the beta subunit is only phosphorylated to a maximum of 1 mol/mol of subunit. With both Mg2+ and Mn2+ the alpha subunit is phosphorylated to a level in excess of 3 mol/mol, a level similar to that obtained for beta subunit phosphorylation in the presence of Mg2+. The support of autophosphorylation by both Co2+ and Ni2+ has characteristics similar to those observed with Mn2+. Although Mn2+ stimulation of autophosphorylation occurs at levels much higher than normal physiological levels, the possible potential of phosphorylase kinase autophosphorylation as a control mechanism is illustrated by the 80- to 100-fold activation that occurs in the presence of Mn2+, a level far in excess of the enzyme activity change normally seen with covalent modification. Autophosphorylation of phosphorylase kinase demonstrates a Km for Mg X ATP2- of 27.7 microM and a Ka for Mg2+ of 3.1 mM. The reaction mechanism of autophosphorylation is intramolecular. This latter observation may indicate that phosphorylase kinase autocatalysis could be of potential physiological relevance and could occur with equal facility in cells containing either constitutively high or low levels of this enzyme.

Adenylate Cyclase in Skeletal Muscle: KINETIC PROPERTIES AND HORMONAL STIMULATION

Abstract Adenylate cyclase was studied in plasma membranes prepared from rabbit skeletal muscle. Such preparations represented an increase in specific activity of 10- to 20-fold over the whole homogenate with a yield of activity of approximately 30%. Various parameters, such as phase contrast microscopy, marker enzyme activities, and chemical composition, suggested that the preparation constituted sarcolemma in a high degree of purity. The Km for substrate (MgATP) was 0.3 to 0.5 mm. The Ka for Mg2+ was 3 to 5 mm. Binding of Mg2+ to a second site resulted in increased reactivity of the catalytic site for substrate. Stimulation by fluoride resulted from an increase in maximal velocity; the Ka for Mg2+ and the Km for substrate were not appreciably altered. The effect of F- was markedly temperature-sensitive and partially irreversible. Fluoride-stimulated activity was particularly sensitive to inhibition by pyrophosphate and this inhibition was competitive with respect to ATP (Ki, 0.45 mm). Catecholamines stimulated the enzyme in a typical β adrenergic fashion. The prominent kinetic effect of epinephrine was (like F-) to increase reaction velocity without affecting affinity for Mg2+ or ATP. The regulation of adenylate cyclase in skeletal muscle may be classified as a V allosteric system since metal ions, F-, and epinephrine all result in increased maximal velocity of the enzyme reaction.

Pressure Effects on Catalysis and Control of Catalysis by Liver Fructose Diphosphatase from an Off-Shore Benthic Fish

SYNOPSIS: At low temperature (2°C), in the absence of FDP and Mg2+, the enzyme fructose disphosphatase (FDPase), extracted from the liver of an off-shore benthic Coryphaenoides species, is inactivated by exposures to relatively low pressures. The substrate, FDP, and the cofactor, Mg2+, protect against this inactivation, so that catalysis per se is not retarded by pressure. In contrast, at alkaline pH, pressure dramatically accelerates the catalytic rate when FDP and Mg2+ are saturating. The volume change of activation, Δ V *, for Coryphaenoides FDPase under these conditions is about −40 cm3/mole. At low concentrations of FDP and saturating concentrations of cofactor, the reaction rate at alkaline pH is pressure-independent. Similarly, at low concentrations of Mg2+ but saturating concentration of FDP, the reaction rate is pressure-independent. The Km for FDP does not change measureably with pressure, while the Ka for Mg2+ increases slightly with pressure. Under conditions of low (probable physiological) FDP and Mg2+ concentrations, it is evident that the reaction rate is determined by the kinetic characteristics of the enzyme and not by its energy-volume relationships, a situation which would appear to be of functional and selective significance to an organism living under constantly high hydrostatic pressure. AMP is a potent specific inhibitor of Coryphaenoides FDPase. The K4 for AMP is essentially pressure-independent both at neutral and alkaline pH, suggesting that efficiency of AMP control of this enzyme is comparable at all pressures likely to be encountered in nature.

The Adaptation of Enzymes to Pressure I. A Comparison of Trout Liver Fructose Diphosphatase with the Homologous Enzyme from an Off-Shore Benthic Fish

SYNOPSIS. At low temperature (3°C), in the absence of substrate and cofactor, trout liver fructose diphosphatase (FDPase) is inactivated by exposures to relatively low pressures. FDP and Mg2+ protect against this inactivation; hence, maximum catalysis at pH 7.5 is pressure insensitive, while at more alkaline pH, it is markedly accelerated by pressure. The volume change of activation, Δ V *, at saturating FDP and Mg2+ concentrations is about −40 cm3/mole. The apparent Km for FDP and the Ka for Mg2+ are markedly increased by pressure. At low FDP or Mg2+ levels these kinetic properties outweigh Δ V * in determining the reaction rate; hence, under these conditions, piessure retards catalysis. Similarly, the K4 for AMP is notably pressure sensitive. Comparable effects of pressure on the kinetic constants for liver FDPase from benthic Corypliaenoides are much less pronounced, suggesting that in these off-shore species enzyme-substrate, enzyme-cofactor, and enzyme-modulator interactions have been tailored through evolution for pressure independent catalytic function.

Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase.

1. The properties of fructose diphosphatase from liver of South American lungfish (Lepidosiren paradoxa) were examined. 2. Saturation curves for substrate (fructose diphosphate) and both cofactors (Mn(2+) and Mg(2+)) are sigmoidal and Hill plots of these results suggest about 2 interacting substrate and cofactor sites/molecule of enzyme. 3. Mn(2+) is an efficient positive modulator of the enzyme and K(a) for Mn(2+) is about 20-30-fold lower than the K(a) for Mg(2+). 4. Lungfish fructose diphosphatase is inhibited by low concentrations of AMP, and the affinity of the enzyme for AMP is insensitive to temperature. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be dependent on temperature, whereas affinity for Mg(2+) is temperature-independent. 6. The pH optimum of the enzyme depends on the presence of the particular cofactor. As pH increases, the K(a) values of both cations are lowered, maximum velocities are increased and the saturation curves for cofactor become hyperbolic. 7. The possible roles of these ions, pH and substrate in the modulation of fructose diphosphatase and gluconeogenic activity in the lungfish are discussed in relation to aestivation and temperature adaptation.