Affinity Labeling of Oxaloacetate Decarboxylase by Novel Dichlorotriazine Linked α-Ketoacids

The 4-aminophenyloxanilic acid and β-mercaptopyruvic acid linked to the reactive diclorotriazine ring, were studied as active site-direct affinity labels towards oxaloacetate decarboxylase (EC 4.1.1.3, OXAD). Oxaloacetate decarboxylase when incubated with 4-aminophenyloxanilic-diclorotriazine (APOD) or β-mercaptopyruvic-diclorotriazine (MPD) at pH 7.0 and 25°C shows a time-dependent and concentration-dependent loss of enzyme activity. The inhibition was irreversible and activity cannot be recovered either by extensive dialysis or gel-filtration chromatography. The enzyme inactivation following the Kitz & Wilson kinetics for time-dependent irreversible inhibition. The observed rate of enzyme inactivation (k _obs) exhibits a non-linear dependence on APOD or MPD concentration with maximum rate of inactivation (k ₃) of 0.013 min^?1 and 0.0046 min^?1 and K _D equal to 20.3 and 156 μM respectively. The inactivation of oxaloacetate decarboxylase by APOD and MPD is competitively inhibited by OXAD substrate and inhibitors, such as oxaloacetate, ADP and oxalic acid whereas Mn⁺² enhances the rate of inactivation. The rate of inactivation of OXAD by APOD shows a pH dependence with an inflection point at 6.8, indicating a possible histidine derivatization by the label. These results show that APOD and MPD demonstrate the characteristics of an active-site probe towards the oxaloacetate binding site of oxaloacetate decarboxylase.     

Affinity labeling of oxaloacetate decarboxylase by novel dichlorotriazine linked alpha-ketoacids

The 4-aminophenyloxanilic acid and -mercaptopyruvic acid linked to the reactive diclorotriazine ring, were studied as active site-direct affinity labels towards oxaloacetate decarboxylase (EC 4.1.1.3, OXAD). Oxaloacetate decarboxylase when incubated with 4-aminophenyloxanilic-diclorotriazine (APOD) or -mercaptopyruvic-diclorotriazine (MPD) at pH 7.0 and 25°C shows a time-dependent and concentration-dependent loss of enzyme activity. The inhibition was irreversible and activity cannot be recovered either by extensive dialysis or gel-filtration chromatography. The enzyme inactivation following the Kitz & Wilson kinetics for time-dependent irreversible inhibition. The observed rate of enzyme inactivation (k _obs) exhibits a non-linear dependence on APOD or MPD concentration with maximum rate of inactivation (k ₃) of 0.013 min^–1 and 0.0046 min^–1 and K _D equal to 20.3 and 156 M respectively. The inactivation of oxaloacetate decarboxylase by APOD and MPD is competitively inhibited by OXAD substrate and inhibitors, such as oxaloacetate, ADP and oxalic acid whereas Mn⁺² enhances the rate of inactivation. The rate of inactivation of OXAD by APOD shows a pH dependence with an inflection point at 6.8, indicating a possible histidine derivatization by the label. These results show that APOD and MPD demonstrate the characteristics of an active-site probe towards the oxaloacetate binding site of oxaloacetate decarboxylase.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

Cations in component reactions of `malic'' enzyme catalysis

The ;malic' enzyme (EC 1.1.1.40) has been purified (300-fold) from wheat germ and its abilities to catalyse the decarboxylation and the hydrogenation of oxaloacetic acid and oxaloacetate esters was studied. The free 1-carboxyl group is essential for the interaction of oxaloacetates and substituted oxaloacetates with the enzyme via cations. The free 4-carboxyl group is required for the decarboxylation but is not indispensable for the hydrogenation. At high concentrations, cations inhibit the enzymic hydrogenation of oxaloacetic acid but not that of 4-ethyl oxaloacetate. A plausible inhibitory mechanism is proposed.     

Kinetic analysis of the reaction mechanism of oxaloacetate decarboxylase from Klebsiella aerogenes

The mechanism of oxaloacetate decarboxylase of Klebsiella aerogenes was investigated by enzyme kinetic methods. The activity of the decarboxylase was strictly dependent on the presence of Na+ or Li+ ions. For Li+ the Km was about 17 times higher and the Vmax about 4 times lower than for Na+. No activity was detectable at Na+ concentrations less than 5 microM. The curve for initial velocity versus Na+ concentration was hyperbolic. Initial velocity patterns with oxaloacetate or Na+ as the varied substrate at various fixed concentrations of the cosubstrate produced a pattern of parallel lines which is characteristic for a ping-pong mechanism. Product inhibition by pyruvate was competitive versus oxaloacetate and noncompetitive versus Na+. Oxalate, a dead-end inhibitor, was competitive versus oxaloacetate and uncompetitive versus Na+. The inhibition patterns are not consistent with a ping-pong mechanism comprising a single catalytic site but are analogous to kinetic patterns observed with the related biotin enzyme transcarboxylase, for which a catalytic mechanism at two different and independent sites has been demonstrated. The kinetic and other data support an oxaloacetate decarboxylase mechanism at two different sites of the enzyme with the intermediate formation of a carboxybiotin-enzyme complex. The first site is the carboxyltransferase which is localized on the alpha chain and the second site is the carboxybiotin-enzyme decarboxylase which is probably localized on the beta and/or gamma subunit. Binding studies with oxalate indicated that this is bound with high affinity to the alpha chain. The affinity was not affected by Na+ or by complex formation with the beta and gamma subunits. Oxalate protected the decarboxylase from heat inactivation but not from tryptic hydrolysis. The carboxybiotin-enzyme intermediate prepared from oxaloacetate decarboxylase with high specific activity was rapidly decarboxylated in the presence of Na+ ions alone. The effect of pyruvate on this reaction, noted previously, probably results from inhomogeneity of the enzyme preparation used which contained a considerable amount of free alpha subunits.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

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 Km values of tag-free enzyme for phosphoenolpyruvate, GDP and bicarbonate were 450, 79 and 20,600 microM, respectively, while those for oxaloacetate and GTP were 4 and 23 microM, 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 Km 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.     

NADP-specific isocitrate dehydrogenase of Mycobacterium phlei ATCC 354: purification and characterization

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) from Mycobacterium phlei ATCC 354 was purified to homogeneity by ammonium sulphate fractionation, followed by DEAE cellulose and Sephadex G-200 chromatography. The pH optimum of the enzyme was 8.5. The Km values for isocitrate and NADP were 74 and 53 microM, respectively. Mn2+ was essential for enzyme activity. The enzyme lost all activity on incubation at 70 degrees C for 15 min; isocitrate and NADP protected against this thermal inactivation. p-Chloromercuribenzoate inhibited the enzyme; pre-incubation of enzyme with isocitrate + Mn2+ prevented this inhibition. The purified enzyme showed concerted inhibition by glyoxylate + oxaloacetate and was inhibited by oxalomalate.     

pH dependence of kinetic parameters for oxalacetate decarboxylation and pyruvate reduction reactions catalyzed by malic enzyme

Both chicken liver NADP-malic enzyme and Ascaris suum NAD-malic enzyme catalyze the metal-dependent decarboxylation of oxalacetate. Both enzymes catalyze the reaction either in the presence or in the absence of dinucleotide. The presence of dinucleotide increases the affinity of oxalacetate for the chicken liver NADP-malic enzyme, but this information could not be obtained in the case of A. suum NAD-malic enzyme because of the low affinity of free enzyme for NAD. The kinetic mechanism for oxalacetate decarboxylation by the chicken liver NADP-malic enzyme is equilibrium ordered at pH values below 5.0 with NADP adding to enzyme first. The Ki for NADP increases by a factor of 10 per pH unit below pH 5.0. An enzyme residue is required protonated for oxalacetate decarboxylation (by both enzymes) and pyruvate reduction (by the NAD-malic enzyme), but the beta-carboxyl of oxalacetate must be unprotonated for reaction (by both enzymes). The pK of the enzyme residue of the chicken liver NADP-malic enzyme decreases from a value of 6.4 in the absence of NADP to about 5.5 with Mg2+ and 4.8 with Mn2+ in the presence of NADP. The pK value of the enzyme residue required protonated for either oxalacetate decarboxylation or pyruvate reduction for the A. suum NAD-malic enzyme is about 5.5-6.0. Although oxalacetate binds equally well to protonated and unprotonated forms of the NADP-enzyme, the NAD-enzyme requires that oxalacetate or pyruvate selectively bind to the protonated form of the enzyme. Both enzymes prefer Mn2+ over Mg2+ for oxalacetate decarboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro studies of the mechanism of inhibition of rat liver uroporphyrinogen decarboxylase activity by ferrous iron under anaerobic conditions

Human porphyria cutanea tarda (PCT) is an unusual consequence of common hepatic disorders such as alcoholic liver disease and iron overload, where hepatic iron plays a key role in the expression of the metabolic lesion, i.e., defective hepatic decarboxylation of porphyrinogens. In this investigation, kinetic studies on a partially purified rat liver uroporphyrinogen decarboxylase have been conducted under controlled conditions to determine how iron perturbs porphyrinogen decarboxylation in vitro. The enzyme, assayed strictly under anaerobic conditions in the dark, was inhibited progressively by ferrous iron. Approximately 0.45 mM ferrous ammonium sulfate was required to observe about 50% inhibition of enzyme activity measured with uroporphyrinogen I as substrate. We showed that (a) all the steps of enzymatic decarboxylation (octa-, hepta-, hexa-, and pentacarboxylic porphyrinogen of isomer I series) were inhibited by ferrous iron. The inhibition was competitive with respect to uroporphyrinogen I and III substrates; (b) the cations, e.g., Fe3+ and Mg2+, had no effect, whereas sulfhydryl group specific cations and compounds such as Hg2+, Zn2+, p-mercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoate) all inhibited the enzyme; (c) the enzyme could be protected from inhibition by Fe2+ and p-mercuribenzoate by preincubation with pentacarboxylic porphyrinogen, a natural substrate and competitive inhibitor. These data suggest for the first time a direct interaction of ferrous iron with cysteinyl residue(s) located at the active site(s) of the enzyme.     

Properties and function of mitochondrial malate enzyme from bass liver

Malate enzyme (L-malate: NADP+ oxidoreductase oxaloacetate decarboxylating, EC 1.1.1.40) from bass liver mitochondria was purified to over 90% of homogeneity by gel filtration, affinity and ion exchange chromatographies. The apparent molecular weight estimated by gel filtration was 316,000. Analysis of the enzyme on sodium dodecylsulphate-polyacrylamide disc gel electrophoresis was shown to be a tetramere protein. The enzyme required bivalent cations for catalysis, (Mn2+ or Mg2+) and displayed a narrow pH optimum (8.4-8.6 for Tris-HCl buffer) and was inactivated by p-chloromercuribenzoate. The double reciprocal initial velocity plots of both of the substrates, NADP and malate, were linear and intercepting at a point that suggests a sequential mechanism. Product inhibition studies with NADP and malate as variable substrate are consistent with an ordered Bi-Ter mechanism.     

Purification and characterization of cytosol phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver.

Cytosol PEP carboxykinase has been purified to electrophoretic homogeneity from bullfrog liver homogenate. The enzyme is a single polypeptide chain with a molecular weight of approximately 72,000-75,000. The purified enzyme catalyzed oxaloacetate decarboxylation (nucleoside triphosphate-supported), phosphoenolpyruvate carboxylation, and an exchange reaction between oxaloacetate and [14C]HCO3-in the presence of ITP or CTP. Manganese is absolutely required for the enzyme-catalyzed phosphoenolpyruvate carboxylation, whereas it can be replaced by Mg2+ for the oxaloacetate decarboxylation and the exchange reaction. The optimal pH of each reaction is dependent on the divalent metal ion used. The dependence of the enzyme activity on Mn2+ is markedly different in the phosphoenolpyuvate carboxylation and the oxaloacetate decarboxylation reactions.     

The mitochondrial glycine cleavage system: differential inhibition by divalent cations of glycine synthesis and glycine decarboxylation in the glycine-CO2 exchange

The exchange of glycine carboxyl carbon with CO2 catalyzed by the combination of chicken liver glycine decarboxylase (P-protein) and aminomethyl carrier protein (H-protein) was markedly inhibited by various divalent cations, although extents of inhibition by individual metal ions varied considerably. Cu2+ and Zn2+, at 100 microM, inhibited the reaction almost completely, and the inhibitions by Co2+ and Ni2+ were also significant, while Mg2+ and Mn2+ did not appreciably affect the reaction. The inhibition by Zn2+ was competitive with both bicarbonate and H-protein and non-competitive with glycine. Of the two reactions involved in the glycine-CO2 exchange, decarboxylation of glycine yielding the H-protein-bound aminomethyl moiety was not significantly affected by 100 microM Zn2+ or Cu2+, but carboxylation of the H-protein-bound aminomethyl moiety to form glycine was strongly inhibited by either Zn2+ or Cu2+. Various degrees of inhibition of the glycine-CO2 exchange by other divalent metal ions could also be accounted for by the inhibition of the carboxylation step of the exchange reaction. The primary site of the action of divalent metal ions is likely to be not P-protein but H-protein, and the binding of metal ions with the H-protein-bound intermediate of glycine decarboxylation was assumed to account for the observed marked inhibition.     

Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Properties of oxaloacetate decarboxylase from Veillonella parvula.

Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe.     

Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump.

We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model.     

Deoxyuridine triphosphate nucleotidohydrolase induced by herpes simplex virus type 1. Purification and characterization of induced enzyme

A deoxyuridine triphosphate nucleotidohydrolase (dUTPase) which is induced in KB cells infected with herpes simplex virus type 1 (HSV-1) was purified approximately 175-fold using affinity, hydrophobic, adsorption, and ion-exchange chromatography techniques. Of the nucleoside triphosphates commonly found in DNA and RNA, only dUTP acted as a substrate for the enzyme, and the apparent Km was 4 microM. While the HSV-1-induced dUTPase exhibited activity in the absence of added divalent cations, the activity was stimulated by Mg2+ and inhibited by EDTA. The inhibition caused by EDTA was reversed by Mg2+, Co2+, or Mn2+. The HSV-1-induced dUTPase was also inhibited by hydroxymercuribenzoate and to a lesser degree by pyrophosphate but not by orthophosphate. The molecular weight of the enzyme was estimated to be 53,000, and its isoelectric point was 5.8. The enzyme exhibited maximal activity over the pH range of 6.5-8.5. The enzyme was thermolabile at 45 degrees C, exhibiting a t1/2 of 35 min. The HSV-1-induced dUTPase was distinguishable from the KB dUTPase by its elution pattern on the various chromatography matrixes, isoelectric point, migration in polyacrylamide gels, thermostability, and response to divalent cations.     

The phosphoenolpyruvate carboxykinase of Trypanosoma (Schizotrypanum) cruzi epimastigotes: molecular, kinetic, and regulatory properties

The phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) of the epimastigote form of Trypanosoma (Schizotrypanum) cruzi has been purified to homogeneity. The enzyme is composed of two apparently identical 42,000 +/- 500 subunits, is highly specific for adenine nucleotides, and has a strict requirement of Mn2+ ions for activity; the activation of the enzyme by ionic Mn2+ reveals that one Mn2+ ion required for each 42,000 subunit. Hyperbolic kinetics are observed for all substrates in the carboxylation reaction with Km (phosphoenolpyruvate) of 0.36 +/- 0.08 mM, Km (HCO-3) of 3.7 +/- 0.2 mM, and Km (Mg-ADP) of 39 +/- 1 microM. In the decarboxylation reaction the kinetics with respect to oxalacetic acid are also hyperbolic with a Km of 27 +/- 3 microM, but towards Mg-ATP there is a biphasic response: hyperbolic at low (less than 250 microM) concentrations with a Km of 39 +/- 1 microM, but at higher concentrations the nucleotide produces a strong inhibition of the enzyme activity. This inhibition is also observed with Mg-GTP and Mg-ITP which are not substrates of the reaction. The results are consistent with an important regulatory function of the enzyme in the amino-acid catabolism of T. cruzi.     

Critical roles of conserved carboxylic acid residues in pigeon cytosolic NADP+-dependent malic enzyme

Malic enzyme catalyses the reduction of NADP+ to NADPH and the decarboxylation of L-malate to pyruvate through a general acid/base mechanism. Previous kinetic and structural studies differ in their interpretation of the amino acids responsible for the general acid/base mechanism. To resolve this discrepancy, we used site-directed mutagenesis and kinetic analysis to study four conserved carboxylic amino acids. With the D257A mutant, the Km for Mn2+ and the kcat decreased relative to those of the wild-type by sevenfold and 28-fold, respectively. With the E234A mutant, the Km for Mg2+ and L-malate increased relative to those of the wild-type by 87-fold and 49-fold, respectively, and the kcat remained unaltered, which suggests that the E234 residue plays a critical role in bivalent metal ion binding. The kcat for the D235A and D258A mutants decreased relative to that of the wild-type by 7800-fold and 5200-fold, respectively, for the overall reaction, by 800-fold and 570-fold, respectively, for the pyruvate reduction partial reaction, and by 371-fold and 151-fold, respectively, for the oxaloacetate decarboxylation. The activities of the overall reaction and the pyruvate reduction partial reaction of the D258A mutant were rescued by the presence of 50 mM sodium azide. In contrast, small free acids did not have a rescue effect on the activities of the E234A, D235A, and D257A mutants. These data suggest that D258 may act as a general base to extract the hydrogen of the C2 hydroxy group of L-malate with the aid of D235-chelated Mn2+ to polarize the hydroxyl group.     

Isocitrate dehydrogenase of Tetrahymena pyriformis

Summary We have studied the isocitrate dehydrogenase ofTetrahymena pyriformis. This enzyme is able to utilize both NAD and NADP, but kinetic studies suggest that the enzymatic activity with NAD is not of physiological significance.Some of the factors that might regulate the NADP-dependent isocitrate dehydrogenase were also studied. This enzyme has an absolute requirement for divalent cations; Mg²⁺ and Mn²⁺ will serve as cofactors but the latter is more effective than the former.It is known that this enzyme is subject to a concerted inhibition by oxaloacetate and glyoxylate. Either glyoxylate or oxaloacetate alone also are capable of inhibiting the enzyme although higher concentrations are required. We have found concerted inhibition also for the NAD-dependent isocitrate dehydrogenase from rat liver and yeast. The activity of theTetrahymena pyriformis enzyme is inhibited by NADPH. This inhibition is competitive with NADP. The Ki and Km values are, respectively, 23µ m and 18µ m.