Isolation and properties of hexokinase from loranthus leaves

Hexokinase was partially purified from the leaves of Dendrophthoe falcata. The optimum pH for the enzyme was 8.5. The enzyme was sensitive to p-CMB and the inhibition could be reversed by 2-mercaptoethanol. The optimum temperature was 40° and energy of activation 6900 cal/mol. The enzyme had an absolute requirement for a divalent metal ion. Although Mg²⁺ was the preferred metal, it could be partially replaced by Mn²⁺ and Ca²⁺. ATP was the most effective phosphoryl donor. Glucose was the best substrate, the K_m values of 0.14 and 0.26 mM were obtained at saturated and sub-saturated ATP concentration. Phosphorylation coefficients show the following order of reactivity of sugars: glucose mannose 2-deoxy D-glucose fructose glucosamine galactose ribose. The K_m value for ATP was 0.16 mM, which increased to 0.35 mM in the presence of 0.5 mM ADP. ADP and 5′-AMP were competitive inhibitors with respect to ATP, and K_i values were 0.4 and 1.2 mM respectively.     

MOUSE NEUROBLASTOMA CELL ADENYLATE CYCLASE: REGULATION BY 2-CHLOROADENOSINE, PROSTAGLANDIN E1 AND THE CATIONS Mg2+, Ca2+ AND Mn

—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg²⁺ or Mn²⁺ in addition to ATP. In the presence of Mg²⁺, the K_m for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn²⁺ cause inhibition. Prostaglandin E₁ and 2-chloroadenosine activate the enzyme. The K_m of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in V_max but does not effect the K_m for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E₁ and 2-chloroadenosine is inversely related to the Mg²⁺ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a K_i of 5 ± 10^?4m . Ca²⁺ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E₁ appears cooperative (i.e. Hill coefficients of ?2). Ca²⁺ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn²⁺ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E₁ of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.     

Decarboxylation of homoarginine and lysine by an enzyme from Lathyrus sativus seedlings

Homoarginine decarboxylase has been purified ca 110-fold from Lathyrus sativus seedlings and resolved from arginine decarboxylase by DEAE-Sephadex column chromatography. The enzyme was less active than arginine decarboxylase and was highly labile. This preparation decarboxylated l-lysine in addition to L-homoarginine. The purified enzyme preparation had an absolute requirement for exogenous Mn²⁺ or Fe²⁺ for both the enzyme activities. The pH and temperature optima for decarboxylation of both homoarginine and lysine were the same viz. 8·4 and 41° respectively. The K_m value l-homoarginine was 3·33 mM and for l-lysine was 0·88 mM. Arginine and homoarginine decarboxylases appear to be different and separable entities having different physico-chemical characteristics, despite the fact that their respective guanido amino acid substrates undergo similar metabolic conversion to guanido- and diamines in this plant system.     

Interaction of Aplysia brasiliana Myoglobin with Mn2+ and Cu2+ ions

Myoglobin of Aplysia brasiliana (MbApB) has been recently purified and characterized and it was shown that the amino acid content is quite different from other myoglobins. A large number of aromatic residues was observed together with the existence of a unique histidine at the proximal heme position. Because of the numerous differences in the amino acid sequence between MbApB and whale myoglobin, it was interesting to investigate the interaction of metal ions like Cu²⁺ and Mn²⁺ with MbApB. In the present work Cu²⁺ complexes with Met-MbApB were studied and show a pH transition between different forms of coordination as revealed by EPR measurements. At high pH the EPR spectrum shows the coordination of the metal to at least four nitrogens from ϵ-NH₃ lysine residues. At lower pH in the range 6.0–9.0 the copper binding site shows a pK change of some of the residues involved in metal coordination. Addition of one equivalent Cu²⁺ per protein does not alter the iron EPR signal. The manganese ion has one binding site in MbApB and a binding constant K_a = ( 11.5 ± 0.8) 10³M⁻¹. The binding of Cu²⁺ to MbApB is stronger than Mn²⁺, K_a^Cu2+ >K_a^Mn2+.     

Purification and some properties of β-N-Acetyl-D-glucosaminidase from viscera of green crab (<Emphasis Type="Italic">Scylla serrata</Emphasis>)

β-N-Acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50°C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45°C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with K_m of 0.424 ± 0.012 mM and V_max of 17.65 ± 0.32 µmol/min at pH 5.8 and 37°C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that Na⁺ and K⁺ have no effects on the enzyme activity; Mg²⁺ and Ca²⁺ slightly activate the enzyme, while Ba²⁺, Zn²⁺, Mn²⁺, Hg²⁺, Pb²⁺, Cu²⁺, and Al³⁺ inhibit the enzyme to different extents.     

The influence of the divalent cations Mn2+ and Mg2+ on the activation of particulate and digitonin-solubilized adenylate cyclase from rat fat cell membranes

A comparison was made between the activation of membrane-bound adenylate cyclase from rat fat cell membranes and the enzyme solubilized with digitonin. The isoprenaline stimulation of the particulate enzyme was enhanced by GTP, both in the presence of Mg²⁺ and Mn²⁺, but no effect of the metal ion nor of GTP was found on the K_a of isoprenaline. The K_a of sodium fluoride for enzyme stimulation was shifted to 3-fold higher concentrations when Mg²⁺ was replaced by Mn²⁺, whereas V decreased. GTP did not influence the K_a of sodium fluoride but reduced V, irrespective of the metal ion. After digitonin solubilization the enzyme was no longer responsive to isoprenaline or GTP; however, V of the sodium fluoride activation was higher in the presence of Mn²⁺ than in the presence of Mg²⁺, and the K_a was found at 15-fold higher concentrations. Both the solubilized and the particulate adenylate cyclase were inhibited by adenosine; this inhibition was also seen with the fluoride stimulated enzyme. We conclude that solubilization with digitonin did not result in an enzyme preparation which preferentially turns over MnATP²⁺, although the fat cell adenylate cyclase possesses a metal ion regulatory site with a higher affinity for Mn²⁺ than for Mg²⁺. The data suggest that the guanyl nucleotide regulatory site and the sodium fluoride-sensitive site are located on different subunits while there is an interaction between the metal ion regulatory site and the fluoride-sensitive site.     

Purification and some properties of discadenine synthase from Dictyostelium discoideum

The enzyme discadenine synthase, which transfers the 3-amino-3-carboxypropyl residue of S-adenosylmethionine to an acceptor, N⁶-isopentenyladenine, from the fruiting body of the cellular slime mold Dictyostelium discoideum, was purified 420-fold. Its apparent molecular mass was 82 000 Da and the isoelectric point was pH 5.8. The K_m value for S-adenosylmethionine was 1.85 · 10⁻⁵ M and for benzyladenine was 7.0 · 10⁻⁷ M. In contrast to theenzyme which catalyzes the formation of 3-(3-amino-3-carboxypropyl)uridine in tRNAs, the present enzyme did not require Mg²⁺ and was not stimulated by ATP. Some other metal ions (Zn²⁺, Mn²⁺, Ca²⁺) showed inhibition at 10–100 mM.     

Purification and properties of isocitrate lyase from Candida brassicae E-17

Isocitrate lyase (EC 4.1.3.1) was purified from acetate-grown cells of Candida brassicae E-17, by ammonium sulfate fractionation and DEAE-cellulose and Sephadex G-200 gel filtration column chromatographies. The purified enzyme was electrophoretically homogeneous. The molecular weight of this enzyme was 290,000 by gel filtration, and it was composed of four identical subunits whose molecular weights were 71,000 each. The pH and temperature optima were 6.8 and 37°C, respectively. The enzyme was stable from pH 6.0 to 7.0. The enzyme was activated by Mg²⁺ and the maximum activity was obtained with a concentration of 8 mM Mg²⁺. The enzyme was also activated by Mn²⁺ and Ba²⁺. The activity of this enzyme was stimulated by reducing agents. The K_m values for dl-isocitrate were 1.5 mM in sodium phosphate buffer and 0.62 mM in imidazole-HCl buffer.     

Purification and properties of dihydroxyacetone kinase from Gluconobacter suboxydans

Different carbon and nitrogen sources had little effect on the level of dihydroxyacetone kinase formed in the cells of Gluconobacter suboxydans. The enzyme was purified to homogeneity from cell-free extract of the organism by ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200 (60-fold purification, 6% yield). Its molecular weight was 260,000; it was stabilized by addition of ATP, dithiothreitol, 2-mercaptoethanol or EDTA, and it reacted optimally at pH 6.5. d-Glyceraldehyde was equally as effective as DHA as a phosphate acceptor (K_m: 0.30 mM each). UTP showed 15% of the reactivity of ATP as a phosphate donor. K_m values for ATP were 0.33 mM in phosphorylation of dihydroxyacetone and 0.39 mM with d-glyceraldehyde. The enzyme activity was dependent on Mg²⁺ but not on Mn²⁺. The reaction with dihydroxyacetone as an acceptor was inhibited by d-glyceraldehyde. The inhibition was competitive with respect to dihydroxyacetone 3K_i=0.09 mM) and noncompetitive with respective to ATP (K_i=5.7 mM).     

Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266?×?10³ units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25?kDa. They showed temperature optima at 50°C and pH optima at 5.0.?km for SPases was reported to be 1.05?mM and 0.186?mM for guaiacol and H₂O₂ respectively. The V_max/K_m value for o-dianisidine was 449 while for H₂O₂ it was 5?×?10⁵. Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5???M) but ferulic acid inhibits its activity at a much lower concentration (0.02???M). Enzymes were stimulated by metal cations like Cu²⁺, Ca²⁺ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn²⁺ and Mg²⁺. Alanine stimulated the enzyme activity (up to 33%; at 0?C100???M) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13?C57% at 0?C100???M).     

Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium

The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn²⁺, Mg²⁺, Co²⁺, and Zn²⁺ shift the optimal pH of phosphatase activity to 6.5 while Be²⁺ and Ca²⁺ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me²⁺ ions, Mn²⁺, Co²⁺, Zn²⁺, and Fe²⁺ decreased the K_m of histidinol phosphate at pH 6.5 from 5.5 mm (without Me²⁺) to 0.14 mm. Ni²⁺ and Be²⁺ increased the K_m to 22.2 and 25.0 mm, respectively, and Ca²⁺ and Mg²⁺ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn²⁺. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn²⁺ to the enzyme before the addition of the substrate, histidinol phosphate. A k_imn²⁺ of 4.3 μm was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me²⁺ ions, Mg²⁺ being the most efficient followed by Mn²⁺. Prolonged incubation with EDTA led to irreversible inactivation.     

Purification and properties of the arginase from Jerusalem artichoke tubers

Arginase [l-arginine amidinohydrolase] in Jerusalem artichoke tubers occurs in a particulate fraction from which it was released in active form by detergent treatment. The particulate enzyme was purified 450-fold with ca 3% yield. The enzyme has a MW of ca 140 000 and pI of 5.3. The enzyme required Mn²⁺ for activity and was unstable when Mn²⁺ was removed. In tissue extracts the K_m for arginine was ca 1OmM, but when purified the K_m (arginine) was 145 mM. The artichoke arginase was shown to be more substrate specific than other plant and animal arginases which have been described, and to be very sensitive to competitive inhibition by indospicine, ornithine and citrulline.     

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

Characterization of Mg2+-ATPase from sheep kidney medulla: magnetic resonance and kinetic studies.

Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg²⁺ or Mn²⁺ indicate a biphasic response to divalent cations. Apparent K_m values of 23 μm for free Mg²⁺ and 3.3 μm for free Mn²⁺ are obtained at low levels of added metal, while K_m values of 0.50 mm for free Mg²⁺ and 0.43 mm for free Mn²⁺ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg²⁺-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent K_m values for free ATP of 6.0 and 1.4 μm with Mg²⁺ and Mn²⁺, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that K_m values for free ATP of 8.0 and 2.0 mm are obtained for Mg²⁺ and Mn²⁺, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg²⁺-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with K_i values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn²⁺ per 469,000 g of protein. The Mn²⁺ binding studies yield a K_D for Mn²⁺ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn²⁺ at low Mn²⁺ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn²⁺ per single high affinity Mn²⁺ site. The K_D for Mn²⁺ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn²⁺ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn²⁺ in the presence of the enzyme is also consistent with the tight binding of a single Mn²⁺ ion per 469,000 M_r protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn²⁺ at the single tight site, ?_b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn²⁺ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10^?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg²⁺-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg²⁺-ATPase described here are discussed.     

Isopentenyl pyrophosphate isomerase from pig liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) from pig liver has been purified 197-fold. The preparation was estimated to contain less than 10% of contaminating protein. The molecular weight determined by gel filtration was 82,500 ± 3,000 and the isoelectric point from isoelectric focusing was in the range 6.0–6.2. N-terminal analysis showed the presence of both leucine and proline. The pH optimum of the enzyme preparation was 6.3. After dialysis against EDTA, activity was restored by either Mn²⁺ or Mg²⁺, the former being more effective. At the optimum pH and concentration of Mn²⁺, K_m and V were 2.7 μm and 6.7 μmol min^?1 mg^?1, respectively. The enzyme was partially inhibited by a variety of terpene mono- and pyrophosphate esters, by inorganic phosphate ions, and by acetate ions; essentially complete inhibition by sulfhydryl-blocking reagents was observed. ATP partially inhibited, the degree of inhibition showing a sigmoid dependence on ATP concentration. Monothiols and dithiothreitol activated the enzyme, as did mevalonic acid.     

Alkaline phosphatase from the excretory system of the grasshopper,Poekilocerus bufonius

Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius was purified with ammonium sulphate fractionation and chromatography on Bio-Gel A-0.5 m. The specific activity of the enzyme is 152 units/mg of protein. The enzyme is a tetramer and the M_r value of the subunit is 72,000 ± 2500 as shown by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 9.6 and an apparent K_m value of 0.28 × 10⁻³ M. The activity of the enzyme reached a maximum at 75°C and the enzyme showed stability at 65°C. The enzyme was inhibited by Ca²⁺, Na⁺ and Fe³⁺ and was stimulated by Zn²⁺, Mn²⁺ and Mg²⁺.     

Effect of hormonal and neuronal agents on adenylate cyclase from smooth muscle of the gastric antrum

Smooth muscle adenylate cyclase of a membrane preparation of canine gastric antrum has been characterized, and the effect of hormonal and neuronal agents examined. The enzyme is active in the presence of Mg²⁺ or Mn²⁺, but is inhibited by Ca²⁺. The K_m is 0.5 mM ATP, similar to the K_m of skeletal muscle adenylate cyclase. The enzyme is activated by isoproterenol but not norepinephrine, consistent with a β₂-catecholamine receptor-adenylate cyclase interaction. Secretin activates the enzyme in concentrations as low as 1 · 10^?11 M, while glucagon was effective only at 1 · 10^?6 M. Prostaglandin E₁ and E₂ have a biphasic effect with activation of adenylate cyclase at 1 · 10^?5 M and a small but significant inhibition of enzyme activity at 1 · 10^?11 M.     

Properties and partial purification of mevalonate kinase from Agave americana

Mevalonate kinase activity was demonstrated in acetone powder extracts from Agave americana leaves, flowers and scape. ATP was the most effective phosphate donor. The enzyme had an optimum pH of 7.9 in Tris-HCl buffer. Dialysis decreased the ability to phosphorylate mevalonic acid (MVA). Partially purified mevalonate kinase reached maximum activity in the presence of 2 mM Mn²⁺ or 6–8 mM Mg²⁺. Higher concentrations of Mn²⁺ were inhibitory, whereas higher concentrations of Mg²⁺ produced only a small decrease in the activity. The amount of mevalonate-5-phosphate (MVAP) formed depended on protein concentration and incubation time. During short incubations, the MVAP formed increased as protein concentration rose, whereas during prolonged incubations (1–6 hr), there was a decrease in the MVAP formed when a certain amount of protein was exceeded. It is suggested that MVAP formed was hydrolysed by a phosphatase present in the extracts. This interfering activity was eliminated when mevalonate kinase is partially purified. The apparent K_m values of the enzyme from leaves were 0.05 mM for MVA and 0. 14 mM for ATP. Similar K_m values are obtained with partially purified mevalonate kinase. The enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 filtration and DEAE-Sephadex A-50 fractionation.     

Characterization of glutamine synthetase in the apple

Glutamine synthetase (l -glutamate: ammonia ligase, ADP-forming, EC 6.3.1.2) in bark tissue of the apple (Malus domestica Borkh. cv. Golden Delicious) was partially purified and characterized. The Mn²⁺- and Mg²⁺-dependent activities were maximal at pH 7.2 and 7.5, respectively. The enzyme was almost completely inactivated within two weeks at 0°C. Both Mg²⁺ and β-mercaptoethanol were effective in stabilizing the enzyme during storage. The enzyme was protected from thermal inactivation at 60°C by the addition of Mg²⁺ and ATP. One-tenth mM phenylmercuric acetate inhibited the Mg²⁺-dependent activity by 50%. Equimolar dithiothreitol protected the enzyme from this inactivation. The K_m values of the enzyme were 0.27, 7.35, and 0.69 mM for ATP, glutamate, and NH₂OH, respectively. The constant for NH⁺₄ was an order of magnitude higher in the presence of Mn²⁺ than Mg²⁺. When the amino acids were externally added to the reaction mixtures, the measurement of P_i exhibited a higher degree of enzyme inhibition than the measurement of γ-glutamyl monohydroxamate (GHA). Ten mM histidine inhibited the Mg²⁺- and Mn²⁺-dependent activities by 26 and 45% respectively. Twenty mM aspartate (d,l -form) inhibited the enzyme 30% in the presence of either Mg²⁺ or Mn²⁺. Aspartate (Mg²⁺-dependent) and histidine (Mn²⁺-dependent) inhibited the enzyme competitively with respect to glutamate, the estimated inhibition constants being 17.6 and 1.6 mM, respectively. At 10 mM, amino acids such as tryptophan, arginine, alanine and citrulline inhibited enzyme activity from 1 to 18%. Glutamine stimulated the Mg²⁺-dependent activity 25% at 25 mM when GHA was measured. Glutamine above 32 mM inhibited the enzyme.     

Kinetic characterization of recombinant human cytosolic phosphoenolpyruvate carboxykinase with and without a His10-tag

We report the first kinetic characterization of human liver cytosolic GTP-dependent phosphoenolpyruvate carboxykinase (GTP-PEPCK), which plays a major role in the development of type 2 diabetes in human. In this work two recombinant forms of the enzyme were studied. One form had a His₁₀-tag and the other was His-tag-free, and with one exception, both exhibited similar kinetic properties. When Mn²⁺ was used as the sole divalent cation, the His₁₀-tagged enzyme, but not the His-tag-free enzyme, was increasingly inhibited at Mn²⁺ concentrations greater than 0.7 mM. This inhibition did not pose any problem in kinetic analysis, for within the relevant Mn²⁺ concentration range the His-tagged human PEPCK behaved almost identically to the tag-free enzyme. This property will bring simplicity and speed to purifying and studying multiple structural variants of this important enzyme. Apparent K_m values of tag-free enzyme for phosphoenolpyruvate, GDP and bicarbonate were 450, 79 and 20,600 μM, respectively, while those for oxaloacetate and GTP were 4 and 23 μM, respectively, emphasizing the enzyme's gluconeogenic character. Bicarbonate (> 100 mM) inhibited OAA-forming activity, which was a new observation with a GTP-PEPCK. The apparent K_m for Mn²⁺ in the PEP-forming direction was 30-fold lower than that for the OAA-forming direction. Mn²⁺ and bicarbonate or CO₂ might regulate the enzyme in vivo.