NAD-linked Enzyme Research Articles

What Is the Metabolic Role of Phosphoenolpyruvate Carboxykinase?

The enzyme phosphoenolpyruvate carboxykinase (GTP; EC 4.1.1.32) (PEPCK)2 has the unusual distinction of being very well studied but metabolically misunderstood. As we will document in this minireview, the enzyme has been almost exclusively linked to gluconeogenesis to the point that changes in the levels of PEPCK mRNA or its activity are associated with the control of hepatic glucose output and, more recently, with alterations in life span. That a tissue such as brown adipose tissue, which does not make glucose, has more PEPCK activity on a protein basis than is present in the liver is largely ignored. In addition, all eukaryotes have a gene for both a mitochondrial (PEPCK-M) and cytosolic (PEPCK-C) form of the enzyme. In the livers of most mammals studied to date (including humans), 50% of the total PEPCK activity is PEPCK-M. However, for reasons to be discussed, only PEPCK-C has been studied in any detail in mammals. Thus, the “strange case of PEPCK-M” deserves our attention. This minireview is an attempt to broaden our prospective on the metabolic role of this enzyme by reviewing the body of literature that has accumulated demonstrating that PEPCK plays a key role in a several metabolic processes associated with cataplerosis.

Inhibition kinetics of NAD-linked enzymes by gossypol acetic acid

Effects of gossypol on human spermatozoal enzymes were investigated. This compound was found to be a potent inhibitor of the NAD-linked enzymes, glucelaldehyde-3-phosphate dehydrogenase (GA3PDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). MDH was inhibited by gossypol when the reaction was carried out in malate-oxalacetate (direct) or oxalacetate-malate (reverse) directions. The I50 of gossypol for the direct reaction was 2.9 microM, whereas that for the reverse reaction was 1.2 microM. Reciprocal plots due to Lineweaver-Burk showed that MDH is inhibited in a noncompetitive manner with respect to both reactions. LDH was also inhibited by this compound when pyruvate or alpha-ketobutyrate was used as a substrate. Gossypol was a noncompetitive inhibitor for LDH. The I50 of gossypol for LDH were 9.8 microM and 11.3 microM, when using pyruvate and alpha-ketobutyrate, respectively as substrates. The I50 of gossypol for GA3PDH and ICDH were 110 microM and 2.7 microM, respectively.

Biochemical basis for carbon monoxide tolerance and butanol production by Butyribacterium methylotrophicum

The biochemical mechanisms for growth tolerance to a 100% CO headspace in cultures, and butanol plus ethanol production from CO by Butyribacterium methylotrophicum were assessed in the wild-type and CO-adapted strains. The CO-adapted strain grew on glucose or CO under a 100% CO headspace, whereas, the growth of the wild-type strain was severely inhibited by 100% CO. The CO-adapted strain, unlike the wild-type, also produced butyrate, from either pyruvate or CO. The CO-adapted strain was a metabolic mutant having higher levels of ferredoxin–NAD oxidoreductase activity, which was not inhibited by NADH. Consequently, only the CO-adapted strain can grow on CO because CO oxidation generates reduced ferredoxin which, via the mutated ferredoxin–NAD reductase activity, forms reduced NADH required for catabolism. When the CO-adapted strain was grown at pH 6.0 it produced butanol (0.33 g/l) and ethanol (0.5 g/l) from CO and the cells contained the following NAD-linked enzyme activities (μmol min−1 mg protein−1): butyraldehyde dehydrogenase (227), butanol dehydrogenase (686), acetaldehyde dehydrogenase (82) and ethanol dehydrogenase (129).

Diversity of lactate metabolism in halophilic archaea

D-Lactate is readily used as a substrate for the growth of species of halophilic archaea belonging to the genera Haloferax and Haloarcula. L-Lactate was used by Haloferax species (Haloferax volcanii, Haloferax mediterranei) only when a substantial concentration of the D-isomer was also present in the medium. On the enzymatic level, considerable diversity was found in the lactate metabolism of the different representatives of the Halobacteriaceae. At least three types of lactate dehydrogenases were detected in halophilic archaea. A high level of activity of an NAD-linked enzyme was present constitutively in Haloarcula species, and a low level of activity was also detected in Haloferax mediterranei. NAD-independent lactate dehydrogenases, oxidizing L-lactate and D-lactate with 2,6-dichlorophenol-indophenol as electron acceptor, were detected in all nine species tested, but L-lactate dehydrogenase activity in Halobacterium species was very low, and Haloarcula species, which possess a high level of activity of NAD-linked lactate dehydrogenase, showed very low activities of both NAD-independent D- and L-lactate dehydrogenase. An inducible lactate racemase, displaying an unusually high pH optimum, was found in Haloferax volcanii. Lactate racemase activity was found constitutively in Haloarcula species, but no activity was detected in Halobacterium species and in Haloferax mediterranei.

Choline transport into rat liver mitochondria.

In mammals, oxidation of choline to betaine occurs within the mitochondria. Choline is firstly oxidized to betaine aldehyde by a n FAD-linked choline dehydrogenase (EC 2.2.9.91) situated on the innerside of the inner mitochondrial membrane (1). This enzyme has high activity in liver and kidney of rats (2, 3). Betaine aldehyde is then oxidized to betaine by betaine aldehyde dehydrogenase (EC 1.2.1.8), an NAD-linked enzyme present in the mitochondrial matrix (4). Consequently, in order for choline to be oxidized, it m u s t f i rs t t raverse t h e mitochondrial inner membrane. Our results show that there is a specific transporter for choline in the mitochondrial inner membrane. At physiological concentrations of choline (60-340pM)[2, 51, uptake of radiolabelled choline showed saturable kinetics with a Km of 220pM and a Vmax of 0.4nmol/mg protein /min a t pH 7.0 and 25OC. The saturable transport process showed a linear dependence on membrane potential and was clearly distinct from the non-specific cation diffusion process described by Brown & Brand (6). Hemicholinium-3 is an analogue of choline and is known to inhibit a range of plasma membrane choline transporters (7). It is also a potent competitive inhibitor ( K i , 17pM) of mitochondrial choline transporter.

Evidence against tight channelling of NADH in hepatocytes

Tritiated substrates at tracer levels were incubated with rat hepatocytes plus 10 m ml-lactate, and the yields of tritium in glucose and water, as well as the tritium distribution on C-6 and C-4 of glucose, determined. Substrates of cytosolic type A NAD-linked dehydrogenases showed some preferential labeling of C-6 of glucose (the pathway involving type A malate dehydrogenase), whereas substrates of cytosolic type B NAD-linked dehydrogenases showed some preferential labeling of C-4 of glucose (the pathway involving type B glyceraldehyde-3P dehydrogenase). The results found are consistent with a classical diffusion model of NADH metabolism, and are at odds with the Srivastava hypothesis (based on isolated enzyme studies) which indicated that direct transfer of NADH can occur between many NAD-linked enzymes, but only when they are of opposite (A or B) specificity.

Use of NAD(P)H and flavoprotein fluorescence signals to characterize the redox state of pyridine nucleotides in epididymal bull spermatozoa

The redox behaviour of the NAD(P) system and flavoproteins was registered by simultaneous fluorescence measurements in epididymal bull spermatozoa. The flavoprotein fluorescence signal can nearly exclusively be attributed to an NAD-linked enzyme, alpha-lipoamide dehydrogenase (Em7.4 = -286 mV). A comparison of intact with digitonin-permeabilized spermatozoa revealed that about 50% of the total NAD(P)H fluorescence signal was of mitochondrial origin. Under equilibrium conditions, the midpoint potentials of the NAD(P)H fluorescence signal of both compartments were almost identical (-300 mV). When lactate was present as substrate, 1 mM caffeine increased respiration oxidizing the NAD(P)H system in both mitochondria and cytosol. This indicates a close relationship of the two NAD pools in spermatozoa.

Different types of formaldehyde-oxidizing dehydrogenases in Nocardia species 239: Purification and characterization of an NAD-dependent aldehyde dehydrogenase

Three different dehydrogenases able to oxidize formaldehyde were found in the Gram-positive methylotroph, Nocardia sp. 239: an NAD-dependent aldehyde dehydrogenase (NA-ADH), an NAD- and factor-dependent formaldehyde dehydrogenase (FD-FDH), and a dye-linked aldehyde dehydrogenase (DL-ADH). The ratio of the activities observed for the two NAD-linked enzymes varied with growth conditions: batch-wise grown cells had nearly the same activities for both enzymes; in fed batch-wise grown cells (methanol limitation) only FD-FDH was detected. The latter is clearly involved in formaldehyde oxidation, since the enzyme and the factor were found only in methanol-grown cells and the enzyme is specific for formaldehyde. In contrast, the two aldehyde dehydrogenases may have significance for aldehyde dissimilation in general, since both activities could also be demonstrated in ethanol-grown cells (but not in glucose-grown cells) and higher aldehydes are even better substrates than formaldehyde. NA-ADH was purified to homogeneity. The enzyme seems to be a homotetramer since it showed a relative molecular mass of 200,000 and the denaturated form of 55,000. Other characteristics are as follows: the enzyme showed substrate inhibition for the aldehydes tested; optimal activity was found at pH 9.2; the reverse reaction was not observed; the enzyme was specific for NAD; GSH, K +, or NH 4 + addition did not stimulate formaldehyde oxidation; the order of NAD and substrate addition to the enzyme was not important; several compounds able to block SH groups were inhibitory. Comparison with NAD-linked aldehyde dehydrogenases from Gram-negative bacteria showed that the Nocardia enzyme is distinct from the enzyme of Pseudomonas putida (EC 1.2.1.46) and of Hyphomicrobium X.

Purification and properties of two succinic semialdehyde dehydrogenases from Klebsiella pneumoniae

Two forms of succinic semialdehyde dehydrogenase have been isolated in Klebsiella pneumoniae M5a1. The two enzymes could be separated by filtration on Sephacryl S-300 and their apparent molecular weights were approx. 275,000 and 300,000. The large enzyme is specific for NADP. The smaller enzyme, which is induced by growth on 3-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and gamma-aminobutyrate, has been purified to 96% homogeneity by affinity chromatography. The NAD-linked succinic semialdehyde dehydrogenase was able to use NADP as cofactor. Its induction is coordinated with 3- and 4-hydroxylase, the enzymes which initiate degradation of 3- and 4-hydroxyphenylacetic acid. The NAD-linked form is also induced by exogenous succinic semialdehyde. The large enzyme is specific for NADP and has been isolated from a defective mutant which lacked the activity of the NAD-linked succinic semialdehyde dehydrogenase. Activity and stability conditions and true K m values for substrates and cosubstrates of the two enzymes were determined. Some aspects of the induction of the NAD-linked enzyme participating in the metabolism of 4-hydroxyphenylacetic and gamma-aminobutyrate were studied.

Immunological comparison of subunits isolated from various hydrogenases of aerobic hydrogen bacteria

Polyclonal, monospecific antibodies were produced against the two subunits ( M r 62 000, and M r 31 000), isolated from the membrane-bound hydrogenase of Alcaligenes eutrophus H16. The antibodies (IgG fractions) were purified from crude sera by Protein A-Sepharose CL-4B chromatography. By double immunodif usion assays and tandem-crossed immunoelectrophoresis the large and the small subunit were demonstrated not to be immunologically related. Immunological comparison of these subunits with the four non-identical subunits ( M r 63 000, 56 000, 30 000 and 26 000) of the NAD-linked, soluble hydrogenase from A. eutrophus H16 showed that the subunits of the membrane-bound hydrogenase did not cross-react with any of the antibodies raised against the four subunits of the NAD-linked enzyme and that, vice versa, none of these four subunits cross-reacted with antibodies raised against the two subunits of the membrane-bound hydrogenase. This means that A. eutrophus H16 contains altogether six non-identical immunologically unrelated hydrogenase polypeptides. The membrane-bound hydrogenases were isolated and purified from various aerobic H 2-oxidizing bacteria: A. eutrophus H16, A. eutrophus type strain, A. eutrophus CH34, A. eutrophus Z1, A. hydrogenophilus, Paracoccus denitrificans and strain Cd2/01. All These proteins resembled each other and each consisted of two non-identical polypeptides. A complete separation of these subunits was achieved at high-yield by preparative FPLC gel filtration of three Superose 12 columns connected in series, using SDS and DTT-containing sodium phosphate buffer (pH 7.0). The small subunits of these enzymes turned out to be immunologically closely related to each other; they were either identical or almost identical. The large subunits were also related, but less pronounced. Only the large subunits from Z1 and type strain reacted fully identical with the H16 subunit. Of the two isolated, homogeneous subunits of the membrane-bound hydrogenase from A. eutrophus H16, the amino acid compositions and the NH 2-terminal sequences have been determined. The results confirmed the diversity of the large and the small subunit. Furthermore, for comparison also the NH 2-terminal sequences of the two subunits from the hydrogenase of A. eutrophus CH34 have been analysed.

Glucose flux and the redox state of pyridine dinucleotides in the rat lens

The concentration of alpha-glycerophosphate (GP) appeared to increase and readily reached a new steady state in lenses incubated with KCN or under hyperglucosic condition. This increase can be explained by the change in NADH/NAD ratio under each condition. The relative ratio of pyridine dinucleotides (NADH/NAD) was calculated from equilibrium equations of two NAD-linked enzymes, lactate dehydrogenase (LDH) and alpha-glycerophosphate dehydrogenase (GPDH). The change in NADH/NAD ratio based on biochemical assays correlates well with that estimated from GPDH reaction. This indicates that measurements of pyridine dinucleotides (e.g. in vivo redox fluorometry) can be used to demonstrate lens metabolic status.

Inhibition of the human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases by nonsteroidal anti-inflammatory drugs

A number of nonsteroidal anti-inflammatory drugs are non-competitive or mixed inhibitors of human placental dehydrogenases. Cis - and trans -sulindac sulfide and cis - and trans -sulindac inhibit the NAD-linked enzyme as well or better than they inhibit various cyclooxygenases in vitro . The remainder of the compounds tested are at least one order of magnitude less effective as inhibitors of the 15-hydroxyprostaglandin dehydrogenases than they are as inhibitors of cyclooxygenases. cis - and trans -sulindac sulfide are sufficiently strong inhibitors of the NAD-linked enzyme (K is of 7.8 μM and 6.8 μM respectively) to raise the possibility that they might also inhibit this enzyme in vivo .

Dirofilaria immitis: Comparison of cytosolic and mitochondrial glutamate dehydrogenases

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8–8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and <100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 μ M NADH, the K m for the substrate, α-ketoglutarate, was equivalent for the two enzymes, but the K m for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 μ M NADH, 1 m M ADP approximately doubled and 1 m M ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 m M glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate K m of M-GDH. However, addition of as little as 3m M glutamate reduced M-GDH velocity about 20%. The two glutamate dehydrogenases of D. immitis were found to differ in almost every respect.

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.

Interactions Between Glycine Decarboxylase, the Tricarboxylic Acid Cycle and the Respiratory Chain in Pea Leaf Mitochondria

In pea leaf and potato mitochondria, external NADH oxidation was inhibited by concurrent oxidation of endogenous NADH and succinate. Succinate oxidation was also inhibited by concurrent oxidation of external NADH, but oxidation of endogenous NADH was not. NAD+-depletion studies suggested that glycine decarboxylase and other NAD-linked enzymes competed for available NAD+ within the matrix. However, at both high and low NAD+ levels, only the tricarboxylic acid cycle enzymes and malic enzyme were inhibited during concurrent oxidation with glycine. Measurements of the oxidation-reduction state of matrix NADH suggested that most of the mitochondria in the preparations contained both glycine decarboxylase and the tricarboxylic acid cycle enzymes and that steady-state levels of NADH were maximal with glycine alone as substrate. That is, there was no evidence for two populations of mitochondria being present. Nonetheless, malate stimulated state 4 and rotenone-inhibited O2 uptake in the presence of glycine. We conclude from these results that the priority glycine has as a substrate for leaf mitochondria is due to a priority that electrons from respiratory complex I have over those from complex II and the external NADH dehydrogenase, and the ability of glycine decarboxylase to compete favourably with tricarboxylic acid cycle enzymes for NAD+ in the matrix. Glycine may inhibit oxidation of other NAD-linked substrates by maintaining high matrix NADH/ NAD+ ratios. However, malate plus pyruvate appear to have access to some electron transport that is not accessible to glycine, at least under ADP-limiting conditions.

Inhibition by gossypol of oxidoreductases from Trypanosoma cruzi

The effects of gossypol, a polyphenolic compound isolated from the cotton plant upon six oxidoreductases from cultured epimastigotes of Typanosoma cruzi were studied. Gossypol was a powerful inhibitor of the α-hydroxyacid and malate dehydrogenases, NAD-linked enzymes, and of glutamate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase, NADP-dependent enzymes. The drug did not have an effect on succinate dehydrogenase, a flavoprotein. The K i values with respect to substrate were 0.73, 0.3 and 3.5 μM for α-hydroxyacid, malate and glutamate dehydrogenases, respectively, and 1.1, 0.19 and 7.8 μM with respect to the coenzyme. Inhibition was noncompetitive with respect to substrate and uncompetitive in relation to the coenzyme.

Substrate specificity of three prostaglandin dehydrogenases

Studies on the substrate specificity, k cat/K m, and effect of inhibitors on the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase (9-ketoprostaglandin reductase) indicate that it is very similar to a human brain carbonyl reductase which also possesses 9-ketoprostaglandin reductase activity. These observations led to a comparison of three apparently homogeneous 15-hydroxyprostaglandin dehydrogenases with varying amounts of 9-ketoprostaglandin reductase activity: an NAD- and an NADP-linked enzyme from human placental and an NADP-linked enzyme from rabbit kidney. All three enzymes are carbonyl reductases for certain non-prostaglandin compounds. The placental NAD-linked enzyme, which has no 9-ketoprostaglandin reductase activity, is the most specific of the three. Although it has carbonyl reductase activity, a comparison of the K m and k cat/K m for prostaglandin and non-prostaglandin substrates of this enzyme suggests that its most likely function is as a 15-hydroxyprostaglandin dehydrogenase. The results of similar comparisons imply that the other two enzymes may function as less specific carbonyl reductases.

Malate Oxidation, Rotenone-Resistance, and Alternative Path Activity in Plant Mitochondria

The effect of cyanide and rotenone on malate (pH 6.8), malate plus glutamate (pH 7.8), citrate, alpha-ketoglutarate, and succinate oxidation by cauliflower (Brassica oleracea L.) bud, sweet potato (Ipomoea batatis L.) tuber, and spinach (Spinacia oleracea and Kalanchoë daigremontiana leaf mitochondria was investigated. Cyanide inhibited all substrates equally with the exception of malate plus glutamate; in this case, inhibition of O(2) uptake was more severe due to an effect of cyanide on aspartate aminotransferase. Azide and antimycin A gave similar inhibitions with all substrates. Subsequent addition of NAD had no effect with any substrate. Providing that oxalacetate accumulation was prevented, rotenone inhibited all NAD-linked substrates equally and caused ADP:O ratios to decrease by one-third. Addition of succinate to mitochondria oxidizing malate stimulated oxygen uptake, but adding citrate and alpha-ketoglutarate did not. These results indicate that there is no direct link between malic enzyme and the rotenone- and cyanide-resistant respiratory pathways, and that there is no need to postulate separate compartmentation of malic enzyme and the other NAD-linked enzymes in the matrix.

On the interaction of nucleotides and glycolytic intermediates with NAD-linked alpha-ketoaldehyde dehydrogenase.

The effect of different nucleotides and glycolytic intermediates was tested on the NAD-linked and NADP-linked alpha-ketoaldehyde dehydrogenases involved in the oxidation of methylglyoxal to pyruvate. ATP, GTP, and ADP strongly inhibit the NAD-linked enzyme whereas activity of the NADP-linked enzyme remained unaltered. NADP at a concentration much below its catalytic concentration strongly inhibited the NAD-linked enzyme. This NADP inhibition decreased with decreasing of the pH of the incubation medium. Fructose 1,6-bisphosphate stimulated and glyceraldehyde 3-phosphate inhibited the activity of the NAD-linked enzyme whereas dihydroxyacetone phosphate inhibited NADP-linked activity. The stimulatory effect of fructose 1,6-bisphosphate diminished with lowering of the pH value. The various effects by several key metabolites of the glycolytic pathway indicate a possible physiological role for these enzymes.

Purification and characterization of NAD and NADP-linked alpha-ketoaldehyde dehydrogenases involved in catalyzing the oxidation of methylglyoxal to pyruvate.

Two alpha-ketoaldehyde dehydrogenases, one catalyzing the oxidation of methylglyoxal to pyruvate with NAD and the other with NADP, were isolated from goat liver and happened to be co-purified. Both the enzymes had been extensively purified to the point where only these two enzymes were present. By affinity chromatography on a thiol-Sepharose column, the two enzymes were separated. Molecular weight of both the enzymes was found to be 42,000 by gel filtration in a Sephadex G-200 column. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel revealed that the enzymes are composed of single subunits. Interaction with mercurials indicated the presence of SH group(s) in the active site of the NAD-linked enzyme only.