Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690

An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O(2) as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp. KY 4690, a soil isolate, to an electrophoretically homogeneous state. The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa. The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family. The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5'-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa. Molecular oxygen served as the sole electron acceptor. These results suggest that aldehyde oxidase from Pseudomonas sp. KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum-molybdpterin-cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of [2Fe-2S] clusters per mol of enzyme protein. The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability.     

Purification and properties of nitroalkane oxidase from Fusarium oxysporum.

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium was purified to homogeneity from an extract of Fusarium oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(NO2)CH2CH3 + O2 + H2O leads to OHCCH2CH3 + HNO2 + H2O2. In addition to 1-nitropropane, 3-nitro-2-pentanol, 2-nitropropane, and nitrocyclohexane are good substrates; the enzyme is designated "nitroalkane oxidase" (EC class 1.7.3). The enzyme has a molecular weight of approximately 185,000 and consists of four subunits identical in molecular weight (47,000). Flavin adenine dinucleotide was required for the enzyme activity and could be replaced in part by riboflavin 5'-phosphate. The maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate, and N-ethylmaleimide. The Michaelis constants are as follows: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM; and flavin adenine dinucleotide, 1.33 micrometer.     

Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa.

A microorganism capable of degrading DL-mandelic acid was isolated from sewage sediment of enrichment culture and was identified as Pseudomonas convexa. It was found to metabolize mandelic acid by a new pathway involving 4-hydroxymandelic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid as aromatic intermediates. All the enzymes of the pathway were demonstrated in cell-free extracts. L-Mandelate-4-hydroxylase, a soluble enzyme, requires tetrahydropteridine, nicotinamide adenine dinucleotide phosphate, reduced form, and Fe2+ for its activity. The next enzyme, L-4-hydroxymandelate oxidase (decarboxylating), a particulate enzyme, requires flavine adenine dinucleotide and Mn2+ for its activity. A nicotinamide adenine dinucleotide-dependent, as well as a nicotinamide adenine dinucleotide phosphate-dependent, benzaldehyde dehydrogenase has been resolved and partially purified.     

Purification and characterization of the oxygen-sensitive acetohydroxy acid synthase from the archaebacterium Methanococcus aeolicus.

Acetohydroxy acid synthase (EC 4.1.3.18) of the archaebacterium Methanococcus aeolicus was purified 1,150-fold to homogeneity. The molecular weight of the purified enzyme was 125,000, and it contained only one type of subunit (M(r) = 58,000). The amino-terminal sequence had 46 to 57% similarity to those of the large subunits of the eubacterial anabolic enzymes and 37 to 43% similarity to those of the yeast and plant enzymes. The methanococcal enzyme had a pH optimum of 7.6. The pI, estimated by chromatofocusing, was 5.6. Activity required Mg2+ or Mn2+ ions, thiamine pyrophosphate, and a flavin. Flavin adenine dinucleotide, flavin mononucleotide, and riboflavin plus 10 mM phosphate all supported activity. However, activity was strongly inhibited by these flavins at 0.3 mM. The Michaelis constants for pyruvate, MgCl2, MnCl2, thiamine pyrophosphate, flavin adenine dinucleotide, and flavin mononucleotide were 6.8 mM, 0.3 mM, 0.16 mM, 1.6 microM, 0.4 microM, and 1.3 microM, respectively. In cell extracts, the enzyme was sensitive to O2 (half-life = 2.7 min with 5% O2 in the headspace), but the purified enzyme was less sensitive to O2 (half-life = 78.0 min with 20% O2). Reconstitution of the enzyme with flavin adenine dinucleotide increased the sensitivity to O2. Moreover, in the assay the homogeneous enzyme was rapidly inactivated by O2, and the concentration required for 50% inhibition (I50) was obtained with an atmosphere of 0.11% O2. The methanococcal enzyme has similarities to the eubacterial and eucaryotic enzymes, consistent with the ancient origin of the archaebacterial enzyme.     

Bacterial short-chain acyl-CoA oxidase: production,purification and characterization

An Arthrobacter nicotianae strain has been found to produce an inducible acyl coenzyme A (CoA) oxidase. Nine times more butyryl-CoA oxidase activity, compared to palmitoyl-CoA oxidase, was found in the cell extract. The addition of flavin adenine dinucleotide (FAD) caused an increase in acyl-CoA oxidase activity and thermal stability. The purified enzyme exhibited a relative molecular mass of 50 000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and 100 000 under non-denaturing conditions. Acyl-CoA oxidase from Arthrobacter nicotianae is highly specific towards short-chain fatty acids. The fastest O₂ uptake was observed with butyryl-CoA as substrate. The enzyme is inhibited by silver and mercury salts.To Professor Dr. Helmut Simon for his 65th birthday Correspondence to: H. Sztajer     

Purification and partial characterization of oxalate oxidase from leaves of forage Sorghum (Sorghum vulgare var. KH-105) seedlings

An oxalate oxidase was purified to apparent homogeneity from the leaves of 10-days old seedlings of forage Sorghum (Sorghum vulgare var. KH-105). The enzyme had a Mr of 124 kDa with two identical subunits, an optimum pH of 4.5, optimum temperature of 37 degrees C and activation energy (Ea) of 2.0338 Kcal/mol. The rate of reaction was linear up to 7 min. K(m) value for oxalate was 0.22 mM. The enzyme was stimulated by Cu2+ and inhibited by EDTA, NaCN, diethyldithiocarbamate, Na2SO4, but unaffected by NaCl at 0.1 mM concentration. Although the enzyme was stimulated by flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), UV and visible spectra of the enzyme did not match with that of a flavoprotein. The positive reaction of the enzyme with orcinol-H2SO4 reagent indicated its glycoprotein nature. The superiority of the purified enzyme over earlier reported oxalate oxidases for determination of urinary oxalate has been demonstrated.     

Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.     

Nitroalkane oxidation by streptomycetes.

Crude cell-free extracts of nine strains of Streptomyces tested for nitroalkane-oxidizing activity showed production of nitrous acid from 2-nitropropane, 1-nitropropane, nitroethane, nitromethane, and 3-nitropropionic acid. These substrates were utilized in most strains but to a decreasing extent in the order given, and different strains varied in their relative efficiency of oxidation. p-Nitrobenzoic acid, p-aminobenzoic acid, enteromycin, and omega-nitro-l-arginine were not attacked. d-Amino acid oxidase, glucose oxidase, glutathione S-transferase, and xanthine oxidase, enzymes potentially responsible for the observed oxidations in crude cellfree extracts, were present at concentrations too low to play any significant role. A nitroalkane-oxidizing enzyme from streptozotocin-producing Streptomyces achromogenes subsp. streptozoticus was partially purified and characterized. It catalyzes the oxidative denitrification of 2-nitropropane as follows: 2CH(3)CH(NO(2))CH(3) + O(2) --> 2CH(3)COCH(3) + 2HNO(2). At the optimum pH of 7.5 of the enzyme, 2-nitropropane was as good a substrate as its sodium salt; t-nitrobutane was not a substrate. Whereas Tiron, oxine, and nitroxyl radical acted as potent inhibitors of this enzyme, superoxide dismutase was essentially without effect. Sodium peroxide abolished a lag phase in the progress curve of the enzyme and afforded stimulation, whereas sodium superoxide did not affect the reaction. Reducing agents, such as glutathione, reduced nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate, reduced form, as well as thiol compounds, were strongly inhibitory, but cyanide had no effect. The S. achromogenes enzyme at the present stage of purification is similar in many respects to the enzyme 2-nitropropane dioxygenase from Hansenula mrakii. The possible involvement of the nitroalkane-oxidizing enzyme in the biosynthesis of antibiotics that contain a nitrogen-nitrogen bond is discussed.     

L-malate oxidation by the electron transport fraction of Azotobacter vinelandii

The membrane-bound l-malate oxidoreductase of Azotobacter vinelandii strain O was found to be a flavoprotein-dependent enzyme associated with the electron transport system (R(3)) of this organism. The particulate R(3) fraction, which possessed the l-malate oxidoreductase, carried out the cyanide-sensitive oxidation of l-malate, d-lactate, reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, succinate, cytochrome c, tetramethyl-p-phenylenediamine, and p-phenylenediamine, with molecular O(2) as the terminal electron acceptor. d-Malate was not oxidized, but l-malate was oxidized to oxalacetate. Phenazine methosulfate (PMS), vitamin K(3), K(3)Fe(CN)(6), nitro blue tetrazolium, and dichloroindophenol all served as good terminal electron acceptors for the l-malate oxidoreductase. Cytochrome c was a poor electron acceptor. Extensive studies on the l-malate oxidase and PMS and K(3) reductases revealed that all were stimulated specifically by flavine adenine dinucleotide and nonspecifically by di- or trivalent cations, i.e., Ca(++), Ba(++), Mn(++), Mg(++), Fe(+++), Ni(++), and Al(+++). All these activities were markedly sensitive to ethylenediaminetetraacetate (EDTA). The V(max) values for the l-malate oxidase, PMS, and vitamin K(3) reductases were, respectively, 3.4, 15.1, and 45.5 mumoles of substrate oxidized per min per mg of protein at 37 C. Spectral studies revealed that the Azotobacter R(3) flavoprotein and cytochromes (a(2), a(1), b(1), c(4), and c(5)) were reduced by l-malate. l-Malate oxidase activity was sensitive to various inhibitors of the electron transport system, namely, p-chloromercuriphenylsulfonic acid, chlorpromazine, 2-n-heptyl-4-hydroxyquinoline-N-oxide, antimycin A, and KCN. Minor inhibitory effects were noted with the inhibitors 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, rotenone, and Amytal.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

Molecular and biochemical characterization of a cytokinin oxidase from maize

It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta. Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance. Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented. Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme. The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals. Expression of the oxidase in maize tissues is described.     

Studies on the structure and mechanism of Streptococcus faecium L-alpha-glycerophosphate oxidase

An FAD-containing L-alpha-glycerophosphate oxidase has been purified to homogeneity from Streptococcus faecium. The purified protein exists as a dimer (subunit Mr = 65,000); each subunit contains 1 mol of FAD. The enzyme contains no iron, as determined by atomic absorption spectroscopy. The alpha-glycerophosphate oxidase reacts reversibly with sulfite to form a covalent N(5) adduct; it preferentially binds the anionic form of the native oxidized FAD, and it also stabilizes the p-quinonoid form of 8-mercapto-FAD. The enzyme shows an unusually high reactivity with ferricyanide in the absence of oxygen; however, there is no evidence for any superoxide ion (O2-.) generation under standard assay conditions. Dithionite titrations of the enzyme reveal an unusual pH dependence for the stabilization of the flavin semiquinone; only at pH 8.5 does significant anionic semiquinone accumulate. L-alpha-Glycerophosphate rapidly reduces the enzyme-bound FAD; in addition, a small amount of catalytically insignificant red semiquinone appears under these conditions. The 5-deaza-FAD-reconstituted enzyme is also reduced by substrate, strongly suggesting that a radical mechanism is not involved in the oxidation of alpha-glycerophosphate. Furthermore, nitroethane anion reduces the native enzyme; this observation suggests that an electron transfer mechanism involving a substrate carbanion is possible with this enzyme.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

Interactions between NADPH oxidase and voltage-gated proton channels: why electron transport depends on proton transport

Leukocytes kill microbes by producing reactive oxygen species, using a multi-component enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Electrons pass from intracellular NADPH through a redox chain within the enzyme, to reduce extracellular O2 to O2-. Electron flux is electrogenic, and rapidly depolarizes the membrane potential. Excessive depolarization can turn off electron transport by self-inhibition, but this is prevented by proton flux that balances the electron flux. Although the membrane potential depolarizes by approximately 100 mV during the respiratory burst (NADPH oxidase activity), NADPH oxidase activity is independent of voltage in this range, which permits optimal function and prevents self-inhibition.     

Formation of Hydrogen Peroxide by Group N Streptococci and Its Effect on Their Growth and Metabolism

The formation of hydrogen peroxide by group N streptococci was found to occur through the action of a reduced nicotinamide adenine dinucleotide (NADH) oxidase which catalyzed the oxidation of NADH by molecular oxygen. The enzyme was activated by flavine adenine dinucleotide. Whereas some of the hydrogen peroxide formed was removed through the action of an NADH peroxidase, sufficient accumulated in media to inhibit the growth, respiration, and viability of these organisms. The amount of hydrogen peroxide which accumulated varied among strains, and this variation could be related to differences in the properties of the NADH oxidase present.     

Purification and Characterization of the Pseudomonas multivorans Glucose-6-Phosphate Dehydrogenase Active with Nicotinamide Adenine Dinucleotide

The Pseudomonas multivorans glucose-6-phosphate dehydrogenase (EC 1.1.1.49) active with nicotinamide adenine dinucleotide, which is inhibitable by adenosine-5'-triphosphate, was purified approximately 1,000-fold from extracts of glucose-grown bacteria, and characterized with respect to subunit composition, response to different inhibitory ligands, and certain other properties. The enzyme was found to be an oligomer composed of four subunits of about 60,000 molecular weight. Reduced nicotinamide adenine dinucleotide phosphate, but not reduced nicotinamide adenine dinucleotide, was found to be a potent inhibitor of its activity. The range of concentrations of reduced nicotinamide adenine dinucleotide phosphate over which inhibition occurred was about 100-fold lower than that for adenosine-5'-triphosphate. The data suggest that reduced nicotinamide adenine dinucleotide phosphate may play an important role in regulation of hexose phosphate metabolism in P. multivorans. Antisera prepared against the purified enzyme strongly inhibited its activity, but failed to inhibit the activity of the nicotinamide adenine dinucleotide phosphate-specific glucose-6-phosphate dehydrogenase which is also present in extracts of this bacterium. Immunodiffusion experiments confirmed the results of the enzyme inhibition studies, and failed to support the idea that the two glucose-6-phosphate dehydrogenase species from P. multivorans represent different oligomeric forms of the same protein.     

Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida.

We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specific lipoamide dehydrogenase produced during growth on valine (molecular weight, 49,000). Branched-chain keto acid dehydrogenase required L-valine, oxidized nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, and magnesium chloride. A partially purified preparation catalyzed the oxidation of 2-keto-[1-14C]isovalerate to [14C]carbon dioxide, isobutyryl-coenzyme A, and reduced nicotinamide adenine dinucleotide in equimolar amounts. Both the Km and the Vmax for 2-ketoisovalerate were affected by the addition of L-valine to the assay mixture. However, only the Vmax values for oxidized nicotinamide adenine dinucleotide and coenzyme A were affected when L-valine was present. This suggested that valine acted by affecting the binding of branched-chain keto acids to subunit E1 of the complex.     

High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica

Cytokinin oxidase/dehydrogenase (CKO/CKX) is a flavoenzyme, which irreversibly inactivates cytokinins by severing the isoprenoid side chain from the adenine/adenosine moiety. There are several genes coding for the enzyme in maize (Zea mays). A Z. mays CKO1 cDNA was cloned in the yeast Yarrowia lipolytica to achieve heterologous protein expression. The recombinant ZmCKO1 was recovered from cultures of transformed yeasts and purified using several chromatographic steps. The enzyme was obtained as a homogeneous protein in a remarkably high-yield and its molecular and kinetic properties were characterized. The enzyme showed a molecular mass of 69 kDa, pI was 6.3. Neutral sugar content of the molecule was 22%. Absorption and fluorescence spectra were in accordance with the presence of FAD as a cofactor. Peptide mass fingerprinting using MALDI-MS correctly assigned the enzyme in MSDB protein database. The enzyme showed a relatively high degree of thermostability (T50=55 degrees C for 30 min incubation). The following pH optimum and K(m) values were determined for natural substrates (measured in the oxidase mode): pH 8.0 for isopentenyl adenine (K(m)=0.5 microM), pH 7.6 for isopentenyl adenosine (K(m)=1.9 microM), pH 7.9 for zeatin (K(m)=1.5 microM) and pH 7.3 for zeatin riboside (K(m)=2.0 microM). ZmCKO1, functioning in the oxidase mode, catalyzes the production of one molecule of H2O2 per one molecule of cytokinin substrate. This finding represents clear evidence for the existence of dual enzyme functionality (oxygen serves as a cosubstrate in the absence of better electron acceptors).     

Purification and properties of pyridine nucleotide-independent L-lactate dehydrogenase from Polyporus circinatus

Cell extracts of Polyporus circinatus grown on lactate catalyze the reduction of 2,6-dichlorophenolindophenol by l-lactate without the participation of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. The enzyme has been purified 78-fold and was homogenous by disc gel electrophoresis. The optimal pH was found to be 6.7. The Michaelis constant for l-lactate was 5.9 x 10(-4) M and the oxalate inhibition constant was 1.5 x 10(-4) M. The nature of the prosthetic group is discussed.     

Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.