Plant succinic semialdehyde dehydrogenase: dissection of nucleotide binding by surface plasmon resonance and fluorescence spectroscopy

Recent kinetic studies revealed distinct modes of inhibition of mitochondrial Arabidopsis thaliana succinic semialdehyde dehydrogenase (At-SSADH1) by AMP and ATP. Inhibition of SSADH by ATP may represent an important mechanism of feedback regulation of the GABA shunt by the respiratory chain. Here we used two approaches to investigate the interaction of ATP with At-SSADH1. Cofactor displacement studies based on the reduced fluorescence intensity of free NADH versus that of enzyme-bound NADH revealed that both AMP and ATP decreased NADH-At-SSADH1 complex formation. The competitive inhibitor AMP displaced all bound NADH, while ATP, a noncompetitive inhibitor, could not, even in great excess, release all NADH from its binding site. To assess the effect of ATP on NAD-At-SSADH, we employed surface plasmon resonance to monitor nucleotide binding to immobilized At-SSADH1. For this, we used a Strep-tag II modified derivative of At-SSADH1 (designated ST-At-SSADH1). The tagged enzyme was tightly and reversibly captured by StrepTactin, which was covalently immobilized on a CM5 chip. The binding constants for NAD(+) and ATP were determined from titration curves and were in good agreement with the constants obtained from enzyme kinetics. Surface plasmon resonance measurements confirmed that ATP binds to a site different from the binding site for NAD(+). GTP competed with ATP. However, only ATP increased the dissociation constant of NAD(+) from SSADH. This explains the reduced affinity of NAD(+)/NADH to At-SSADH1 in the presence of ATP, as revealed by enzymatic kinetics, and supports our model of feedback regulation of SSADH and the GABA shunt by ATP.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

Kinetic properties of 5-carboxymethyl-2-hydroxymuconate semialdehyde dehydrogenase from Escherichia coli

Kinetic properties of purified 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMSA) dehydrogenase (EC 1.2.1.-) in the 4-hydroxyphenylacetate meta-cleavage pathway from Escherichia coli have been studied. The temperature--activity relationship for the enzyme from 27 to 45 degrees C showed an Arrhenius plot with an inflexion at 36 degrees C. When 5-carboxymethyl-2-hydroxymuconic semialdehyde and NAD were used as variable substrates, the double reciprocal plots were all linear and the lines intersected at one point below the horizontal axis, suggesting that a sequential mechanism is operating. From the replots of intercepts and slopes against reciprocal substrate concentrations were calculated Km (CHMSA) = 9.0 +/- 1.02 microM, Km (NAD) = 29.1 +/- 4.65 microM and the value for the dissociation constant of enzyme--NAD complex = 6.3 +/- 1.21 microM. ATP and the product of the reaction (NADH) acted as competitive inhibitors of the enzyme with respect to NAD. Apparent Ki values, estimated from Dixon plots, were 25.0 +/- 3.5 and 88.0 +/- 22.1 microM for NADH and ATP, respectively.     

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).     

Succinic semialdehyde dehydrogenase activity in bovine adrenal medulla and blood platelets: a comparative study with the brain enzyme

In the present paper we report the presence of succinic semialdehyde dehydrogenase (SSADH) in bovine adrenal medulla and blood platelets. Both enzymes present some analogies with the brain enzyme in terms of cofactor requirements, optimal pH, mitochondrial localizaton and inhibition by AMP. However, the activity of the platelet enzyme is 100 times lower than that of the brain and affinities of both enzymes for their specific substrate succinic semialdehyde and NAD are different. The presence of SSADH in adrenal medulla and blood platelets allows us to confirm the presence of a complete GABA bypass in these tissues, where the neurotransmitter could have important regulator functions.     

Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex.

1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.     

Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - 100 microM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.     

Salivary apyrase of Rhodnius prolixus. Kinetics and purification.

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5'-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.     

Binding of nucleotides to an extramitochondrial acetyl-CoA hydrolase from rat liver

Cold labile extramitochondrial acetyl-CoA hydrolase (dimeric form) purified from rat liver was activated by various nucleoside triphosphates and inhibited by various nucleoside diphosphates. Activation of acetyl-CoA hydrolase by ATP was inhibited by a low concentration of ADP (Ki congruent to 6.8 microM) or a high concentration of AMP (Ki congruent to 2.3 mM). ADP and AMP were competitive inhibitors of ATP. A Scatchard plot of the binding of ATP to acetyl-CoA hydrolase (dimer) at room temperature gave a value of 25 microM for the dissociation constant with at least 2 binding sites/mol of dimer. Cold-treated monomeric enzyme also associated with ATP-agarose, suggesting that the monomeric form of the enzyme also has a nucleotide binding site(s), probably at least 1 binding site/mol of monomer. Phenylglyoxal or 2,3-butanedione, both of which modify arginyl residues of protein, inactivated acetyl-CoA hydrolase. ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by these reagents, while ADP (an inhibitor) greatly (a substratelike, competitive inhibitor), and CoASH (a product) were less effective. However, addition of ADP plus valeryl-CoA (or CoASH) effectively prevented the inactivation by 2,3-butanedione, but that is not the case for phenylglyoxal. These results suggest that one or more arginyl residues are involved in the nucleotide binding site of extramitochondrial acetyl-CoA hydrolase and that their nucleotide binding sites locate near the substrate binding site.     

Identification of an unusual AT(D)Pase-like activity in multifunctional NAD glycohydrolase from the venom of Agkistrodon acutus

NAD-glycohydrolases (NADases) are ubiquitous enzymes that possess NAD glycohydrolase, ADPR cyclase or cADPR hydrolase activity. All these activities are attributed to the NADase-catalyzed cleavage of C-N glycosyl bond. AA-NADase purified from the venom of Agkistrodon acutus is different from the known NADases, for it consists of two chains linked with disulfide-bond(s) and contains one Cu(2+) ion. Here, we show that AA-NADase is not only able to cleave the C-N glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the phosphoanhydride linkages of ATP, ADP and AMP-PNP to yield AMP. AA-NADase selectively cleaves the P-O-P bond of ATP, ADP and AMP-PNP without the cleavage of P-O-P bond of NAD. The hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. ATP is the excellent substrate for AA-NADase with the highest specificity constant k(cat)/K(m) of 293+/-7mM(-1)s(-1). AA-NADase catalyzes the hydrolysis of ATP to produce AMP with an intermediate ADP. AA-NADase binds with one AMP with high affinity determined by isothermal titration calorimetry (ITC). AMP is an efficient inhibitor against NAD. AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities.     

The cell death factor,cell wall elicitor of rice blast fungus (Magnaporthe grisea) causes metabolic alterations including GABA shunt in rice cultured cells

An elicitor derived from the cell wall of rice blast fungus (Magnaporthe grisea) causes cell death in suspension cultured cells of rice (Oryza sativa L.). To elucidate the role of M. grisea elicitor on metabolic pathway of rice cells, we performed metabolite profiling using capillary electrophoresis-mass spectrometry (CE/MS). Treatment with M. grisea elicitor increased the amounts of antioxidants and free amino acids and decreased the amount of metabolites in the tricarboxylic acid (TCA) cycle. Lower ATP concentration caused aberrant energy charge, concurrently with reduced amount of NAD(P)H in elicitor treated cells. Among free amino acids detected in this study, the level of gamma-aminobutyric acid (GABA) increased. GABA is metabolized through a bypass pathway of the TCA cycle called GABA shunt, which is composed of glutamate decarboxylase (GAD), GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). While M. grisea elicitor negligibly affected GAD and SSADH, GABA-T activity significantly decreased. The decrease in GABA-T activity was recovered by NADPH oxidase inhibitor, which prevents cell death induced by M. grisea elicitor. Thus, GABA accumulation observed in rice cells under elicitor stress is partly associated with GABA-T activity.Key words: metabolome, Magnaporthe grisea, capillary electrophoresis, mass spectrometry, gamma-aminobutyric acid, GABA transaminase, Oryza sativa     

The deposition of calcium pyrophosphate by NTP pyrophosphohydrolase of matrix vesicles from fetal bovine epiphyseal cartilage

About 5 mumol CaPPi/mg protein was deposited within 3 h in the presence of the reaction mixtures containing 1 mM ATP, 2 mM Ca2+, 1 mM Pi, and 17 micrograms of purified NTP pyrophosphohydrolase. At 1 mM ATP, 50% of the deposition was inhibited by 0.5-1 mM of various substrate and product analogues including AMP, ADP, and ethylene hydroxyl diphosphonate. The magnitude of inhibition on NTP pyrophosphohydrolase activity was in the order of AMP = CMP = ADP greater than adenosine greater than adenine greater than NAD = NADP. AMP, CMP, ADP, and adenosine are competitive inhibitors. The modes of inhibition by adenine, NAD, and NADP differ from the competitive inhibition. Ribose, 3'-AMP, 2'-AMP, and cAMP did not inhibit the enzyme activity.     

Functional characterization of a Drosophila melanogaster succinic semialdehyde dehydrogenase and a non-specific aldehyde dehydrogenase

The putative Drosophila (D.) melanogaster gene ortholog of mammalian succinic semialdehyde dehydrogenase (SSADH, EC1.2.1.24; NM_143151) that is involved in the degradation of the neurotransmitter GABA, and the putative D. melanogaster aldehyde dehydrogenase gene Aldh (NM_135441) were cloned and expressed as enzymatically active maltose binding protein (MalE) fusion products in Escherichia coli. The identities of the NM_143151 gene product as NAD+-dependent SSADH and of the Aldh gene product as NAD+-dependent non-specific aldehyde dehydrogenase (ALDH, EC1.2.1.3) were established by substrate specificity studies using 30 different aldehydes. In the case of D. melanogaster MalE-SSADH, the Michaelis constants (K(M)s) for the specific substrates succinic semialdehyde and NAD+ was 4.7 and 90.9 microM, respectively. For D. melanogaster MalE-ALDH the K(M) of the putative in vivo substrate acetaldehyde was 0.9 microM while for NAD+, a K(M) of 62.7 microM was determined. Site-directed mutagenesis studies on D. melanogaster MalE-SSADH suggest that cysteine 311 and glutamic acid 277 of this enzyme are likely candidates for the active site residues directly involved in catalysis.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

CoA-dependent methylmalonate-semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase superfamily. cDNA cloning, evolutionary relationships, and tissue distribution.

Three overlapping cDNA clones encoding methylmalonate-semialdehyde dehydrogenase (MMSDH; 2-methyl-3-oxopropanoate:NAD+ oxidoreductase (CoA-propanoylating); EC 1.2.1.27) have been isolated by screening a rat liver lambda gt 11 library with nondegenerate oligonucleotide probes synthesized according to polymerase chain reaction-amplified portions coding for the N-terminal amino acid sequence of rat liver MMSDH. The three clones cover a total of 1942 base pairs of cDNA, with an open reading frame of 1569 base pairs. The authenticity of the composite cDNA was confirmed by a perfect match of 43 amino acids known from protein sequencing. The composite cDNA predicts a 503 amino acid mature protein with M(r) = 55,330, consistent with previous estimates. Polymerase chain reaction was used to obtain the sequence of the 32 amino acids corresponding to the mitochondrial entry peptide. Northern blot analysis of total RNA from several rat tissues showed a single mRNA band of 3.8 kilobases. Relative mRNA levels were: kidney greater than liver greater than heart greater than muscle greater than brain, which differed somewhat from relative MMSDH protein levels determined by Western blot analysis: liver = kidney greater than heart greater than muscle greater than brain. A 1423-base pair cDNA clone encoding human MMSDH was isolated from a human liver lambda gt 11 library. The human MMSDH cDNA contains an open reading frame of 1293 base pairs that encodes the protein from Leu-74 to the C terminus. Human and rat MMSDH share 89.6 and 97.7% identity in nucleotide and protein sequence, respectively. MMSDH clearly belongs to a superfamily of aldehyde dehydrogenases and is closely related to betaine aldehyde dehydrogenase, 2-hydroxymuconic semialdehyde dehydrogenase, and class 1 and 2 aldehyde dehydrogenases.     

Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.     

Effect of Mg2, ATP and some analogs on phosphorylase phosphatase from adrenal cortex.

The effect of Mg2, ATP and some of its analogs was studied on the spontaneously active and the ATP-Mg-dependent forms of phosphorylase phosphatase extracted from adrenal cortex. Inhibition of the spontaneously active form was observed with Mg2 (Ki - 9mM), ATP (Ki = 9micronM), 2'-doxy-ATP (Ki = 8 micronM), AtetraP (Ki = 9 micronM), AMP(CH2)PP (Ki = 11 micronM), ADP(CH2)P (Ki = 19 micronM), ADP(NH)P (Ki = 16micronM) and ADP (Ki = 25micronM). Activation of the ATP-Mg-dependent form was obtained with Mg2 (Ka = 0.55mM) (to a lower extent) and with ATP (Ka = 2micronM), 2'-deoxy-ATP (Ka = 6micronM) or AtetraP (Ka = 15micronM) in the presence of 0.5mM Mg2. Activation with AMP(CH2)PP was only observed in the presence of high concentrations (5mM) of Mg2 (Ka = 13micronM). No activation at all was observed with ADP(CH2)P or ADP(NH)P. Even though the activation of the ATP-Mg-dependent form does not seem to involve a kinase reaction, the stimulation by ATP or its analogs is rather specific, since it does not occur with analogs in which a methylene group or a nitrogen is substituted for the oxygen between the beta- and gamma-phosphates.     

Inhibition of succinic semialdehyde dehydrogenase activity by alkenal products of lipid peroxidation

Lipid peroxidation causes the generation of the neurotoxic aldehydes acrolein and 4-hydroxy-trans-2-nonenal (HNE). These products are elevated in neurodegenerative diseases and acute CNS trauma. Previous studies demonstrate that mitochondrial class 2 aldehyde dehydrogenase (ALDH2) is susceptible to inactivation by these alkenals. In the liver and brain another mitochondrial aldehyde dehydrogenase, succinic semialdehyde dehydrogenase (SSADH/ALDH5A1), is present. In this study, we tested the hypothesis that aldehyde products of lipid peroxidation inhibit SSADH activity using the endogenous substrate, succinic semialdehyde (SSA, 50 microM). Acrolein potently inhibited SSADH activity (IC(50)=15 microM) in rat brain mitochondrial preparations. This inhibition was of an irreversible and noncompetitive nature. HNE inhibited activity with an IC(50) of 110 microM. Trans-2-hexenal (HEX) and crotonaldehyde (100 microM each) did not inhibit activity. These data suggest that acrolein and HNE disrupt SSA metabolism and may have subsequent effects on CNS neurochemistry.     

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.