Coenzyme A: fatty acid synthetase apoenzyme 4'-phosphopantetheine transferase in yeast

A mixture of two pantetheine-free mutant fatty acid synthetases was dissociated and recombined $\text{in}$$\text{vitro}$ to form a hybrid apoenzyme complex. $\text{In}$$\text{vivo}$ the corresponding $\text{Saccharomyces}$$\text{cerevisiae}$$\text{fas}$-mutants exhibit interallelic complementation when crossed with each other and the enzyme synthesized in the resulting diploid contains pantetheine and exhibits overall fatty acid synthetase activity. Accordingly, the hybrid apoenzyme formed $\text{in}$$\text{vitro}$ could be activated to holo-fatty acid synthetase when incubated with coenzyme A and a partially purified yeast cell extract. The enzyme coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase has thus been identified in yeast. Further studies on the mechanism of fatty acid synthetase holoenzyme formation will now be possible.     

Stimulation of brain aromatic alkylamine N-methyltransferase activity by FAD and methylcobalamin

We studied the effects of methylcobalamin alone and with various reducing systems (FADH₂, FAD or its analogues in combination with NADH or β-mercaptoethanol) on the activity of partially purified aromatic alkylamine N-methyltransferase (AANMT) from rat brain. As expected, the specific activity of AANMT in the presence of methylcobalamin alone was enhanced (125% of control). Surprisingly, in the presence of FAD alone (but not FADH₂$\text{et cetera}$) it was 175% of control; and int the presence of methylcobalamin plus FAD plus β-mercaptoethanol it reached 307% of control. Preliminary evidence suggests that FAD induces allosteric kinetics for the activated enzyme with respect to the methyl donor 5-methyltetrahydrofolic acid (5-MTHF).     

Inactivation of glutamate decarboxylase by bromopyruvate

Bromopyruvate was shown to inhibit $\text{E. coli}$ glutamate decarboxylase competitively with respect to L-glutamate. High concentrations of bromopyruvate caused a time-dependent inactivation of glutamate decarboxylase. However, the apoenzyme was rapidly and irreversibly inactivated by bromopyruvate with an inactivation constant of 490 1 mole^?1 min^?1 at pH 5.7. Studies with labeled bromopyruvate indicated that approximately 1.7 moles of inhibitor were bound per subunit of apoenzyme.     

The role of molybdenum in formation of the NADPH-nitrate reductase by Aspergillus nidulans

Non-nitrate reducing mutants of $\text{Aspergillus}$$\text{nidulans}$ have been noted to produce either a nitrate inducible or constitutive NADPH-cytochrome $\text{c}$ reductase which resides in either a 4.5s or a 7.8s protein. The latter closely resembles the nitrate inducible, FAD dependent NADPH-nitrate reductase from the wild type. Measurement of flavin adenine dinucleotide (FAD) and molybdenum (Mo) in these two proteins revealed significant differences particularly in Mo. The concepts that a nitrate inducible $\text{nia}$ gene product constitutes the major flavin bearing component of the enzyme and that a constitutively produced $\text{cnx}$ gene product is implicated in formation of the larger Mo bearing multimer are further supported.     

Occurrence in mammalian liver of a protein which replaces the B protein of E. coli quinolinate synthetase.

$\text{Escherichia coli}$ contains two proteins (A and B) which together convert dihydroxyacetone phosphate and aspartate to quinolinic acid, a precursor of NAD. Although mammalian liver homogenate does not catalyze this reaction it contains a protein which will replace the B protein of the $\text{E. coli}$ system. The behavior of the liver protein on Sephadex G-75 suggests it is much smaller than the $\text{E. coli}$ B protein. Liver B protein also appears to contain tightly bound FAD while FAD is easily removed from the $\text{E. coli}$ B protein. The pH optimum for the hybrid system $\text{E. coli}$ A protein-liver B protein is 9.0 while in the pure $\text{E. coli}$ system the optimum is pH 8.0. The hybrid system is inhibited by NAD to the same extent as the pure $\text{E. coli}$ system.     

A Study on the Kinetic Mechanism of Apoenzyme Reconstitution from Aerococcus viridans Lactate Oxidase

The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k₃ and k_-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.     

The reduced nicotinamide adenine dinucleotide “oxidase” of Acholeplasma laidlawii membranes

An NADH dehydrogenase possessing a specific activity 3–5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0 % ethanol at 43 °C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1 : 2 FAD to FMN, and 30–40 % lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O₂, ubiquinone $\text{Q}{}_{10}$ or cytochrome $\text{c}$. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0 % ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{'}\text{s}$ with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller $\text{V'}\text{s}$ with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg²⁺, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O₂ uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.     

Nitrate reductase from Penicilliumchrysogenum: Kinetic mechanism at sub-optimum pH

The steady-state kinetics of the NADPH + FAD-dependent reduction of nitrate by nitrate reductase from $\text{Penicillium}\text{chrysogenum}$ was studied at pH 6.18. At this sub-optimum pH, V_max was about 83 units × mg protein^?1 compared with 225 units × mg protein^?1 at pH 7.20. All initial velocity reciprocal plot patterns at pH 6.18 as well as the NADP⁺/nitrate product inhibition pattern were intersecting. In contrast, the NADP(H)/nitrate plots at pH 7.20 were parallel (Renosto, F. $\text{et}\text{al.}$ J. Biol. Chem. $\text{256}$, 8616, 1981). A major effect of lowering the assay pH was to change the K_m for FAD from 0.17 μM at pH 7.20 to 4 μM at pH 6.18. The results suggest that nitrate reductase has a steady-state random kinetic mechanism in which k_cat in the forward direction at pH 7.20 (ca. 375 sec^?1) is greater that k_off for the dissociation of one or more substrates. Several observations suggest that k_off for FAD is extremely small at pH 7.20.     

Activation of Phycomyces adenosine 3', 5'-monophosphate phosphodiesterase by blue light.

Cyclic AMP phosphodiesterase can be extracted from sporangiophores of $\text{Phycomyces}\text{blakesleeanus}$. Activity is enhanced by 1–10 μM FAD and FMN but not by riboflavin. Moderate intensity of blue light also activates the enzyme, especially in the presence of 1mM GTP. The enzyme must be extracted and stored in the absence of blue light for this result. Forty times the intensity of red light has no effect. This finding is consistent with the very sudden transient drop in cyclic AMP level upon light stimulation in the intact sporangiophore.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

Studies on NADPH-cytochrome c reductase I: Isolation and several properties of the crystalline enzyme from ale yeast.

NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min^?1mg^?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 10⁴ was obtained with use of a $\text{V}\text{?}{}_{\mathrm{20\; °}}\text{= 0.741}$ calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 3.4 (± 0.1) × 10⁴ resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 10⁴. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10^?8m for FAD and 4.4 × 10^?8m for FMN.     

Fluorescence energy transfer in tryptophanase

Excitation of apotryptophanase from Escherichia coli$\text{B}\text{lt7-A}$ at 290 nm yielded a fluorescence emission centered at 340 nm. Binding of pyridoxal phosphate to apoenzyme induced quenching of protein fluorescence concomitant with an appearance of another peak at 510 nm by way of energy transfer from tryptophan. Based on the results, an approximate distance between the coenzyme and tryptophan was estimated to be 18–24 Å according to the Förster's theory. The ozone-inactivated enzyme yielded only the 340 nm-peak upon excitation at 290 nm following reconstitution with the coenzyme. The fluorescence decay time of the tryptophyl residue was somewhat increased by ozone-inactivation. These results suggest that the tryptophyl residue essential for the activity is involved in a direct interaction with the coenzyme.     

Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat liver.

NADPH-cytochrome P-450 reductase with capacity to support cytochrome P-450-dependent drug metabolism and to reduce artificial electron acceptors has been purified to apparent homogeneity by solubilization with Renex 690 and chromatography on DEAE-Sephadex, Agarose and QAE-Sephadex. The purified protein migrates as a single band on native and SDS-polyacrylamide gel electrophoresis, exhibits a minimum molecular weight of 80,000 daltons and contains 1 molecule each of FAD and FMN per 80,000 molecular weight. The specific activity for cytochrome $\text{c}$ as electron acceptor is 48.8 μmoles per min and for substrate hydroxylation of benzphetamine measured as NADPH oxidation in the presence of cytochrome P-450 and phosphatidylcholine is 2.5 μmoles per min.     

Distribution and transport of apo- and holocytochrome b5 in the endoplasmic reticulum of rat liver

The transport and distribution of apo- and holocytochrome $\text{b}{}_5$ was investigated with the aid of specific antibodies. The holoenzyme was found to be localized mainly in the rough and smooth endoplasmic reticulum and in the Golgi system but some precipitation could also be obtained in the outer mitochondrial membranes and in the peroxisomes. The apoenzyme, however, could only be detected in the endoplasmic reticulum-Golgi system, which also was shown to be the sole site for incorporation of the prosthetic heme moiety. Time-course studies revealed that the labeled enzyme appeared both as apoenzyme and as holoenzyme in the rough endoplasmic reticulum 10 min after in vivo injection of radioactive leucine and that further transport to the smooth endoplasmic reticulum occurred within 10 min. The subsequent transport to other organelles, however, required a somewhat longer time and peak radioactivity in outer mitochondrial membranes was not attained until after 40 min.     

Reactivity of an FAD-dependent oxygenase with free flavins: a new mode of uncoupling in flavoprotein oxygenases.

Conversion of 2-methyl-3-hydroxypyridine-5-carboxylic acid (Cpd I) to α-(N-acetylaminomethylene)succinic acid (Cpd A) is catalyzed by an FAD protein, Cpd I oxygenase (Sparrow, et al., J. Biol. Chem. [1969] $\text{244}$, 2590–2600) according to the equation: Cpd I + O₂ + NADH + H⁺ → Cpd A + NAD⁺. When free FAD, FMN or riboflavin is added to reaction mixtures, oxidation of NADH remains dependent on presence of oxygenase and Cpd I, but is partially uncoupled from the oxygenation of Cpd I. Under these conditions, free reduced flavins appear in solution, as shown by their ability to reduce cytochrome c. These effects are not due to an increased rate of NADH oxidation. Such uncoupling may lead to appearance of artifactual species of activated oxygen or flavin that play no intermediate role in the oxygenase reaction.     

Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x 10(3) M-1 s-1 at 20 degrees C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.     

Highly purified detergent-solubilized NADPH-cytochrome P-450 reductase from phenobarbital-induced rat liver microsomes

NADPH-cytochrome P-450 reductase was highly purified from liver microsomes of phenobarbital-induced rats by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, and hydroxylapatite in the presence of deoxycholate or Renex 690, a nonionic detergent. The purified enzyme gave a single major band with a molecular weight of 79,000 daltons on SDS-polyacrylamide gel electrophoresis. FMN and FAD were present in about equal amounts. The most active reductase preparation catalyzed the reduction of 40.9 μmoles of cytochrome $\text{c}$ per min per mg of protein and, as an indirect measure of cytochrome P-450 reduction, the oxidation of 2.0 μmoles of NADPH per min per mg of protein in a reconstituted hydroxylation system containing benzphetamine as the substrate.     

Properties of fatty acyl-CoA oxidase from rat liver, a peroxisomal flavoprotein

Fatty acyl-CoA oxidase from rat liver was partially purified and characterized as a peroxisomal flavoprotein oxidase. A sedimentation coefficient of 7.7 S was estimated from sucrose gradients and a Stokes radius of 42.3 Å was deduced from gel-exclusion chromatography. These data allow to estimate a molecular weight of 136,000 and a frictional ratio of 1.1. FAD, specifically required as a prosthetic group, is weakly bound. Still, FAD displays greater affinity for the free ap$\text{o}$ enzyme than for the putative apoenzyme-substrate complex formed with palmitoyl-CoA. In addition, it was established that the subcellular distribution of the fatty acyl-CoA oxidase, in complete liver homogenates fractionated in Metrizamide density gradients, parallels that of the peroxisomal marker catalase.