Oxidation and dehalogenation of 4-chlorophenylacetate by a two-component enzyme system from Pseudomonas sp. strain CBS3

In cell-free extracts from Pseudomonas sp. strain CBS3 the conversion of 4-chlorophenylacetate to 3,4-dihydroxyphenylacetate was demonstrated. By Sephacryl S-200 chromatography two protein fractions, A and B, were obtained which both were essential for enzyme activity. Fe2+ and NADH were cofactors of the reaction. NADPH also activated the enzyme, but less effectively than NADH. FAD had no influence on enzyme activity. 4-Hydroxyphenylacetate, 4-chloro-3-hydroxyphenylacetate, and 3-chloro-4-hydroxyphenylacetate were poor substrates for the enzyme, suggesting that these substances are not intermediates of the reaction. We therefore suggest that the reaction proceeds via a dioxygenated intermediate.     

Selective chemical modification of amino acid residues in the flavin adenine dinucleotide binding site of NADPH-ferredoxin reductase.

1. An apo-NADPH-ferredoxin reductase was prepared from holo-NADPH-ferredoxin reductase (EC 1.18.1.2) from bovine adrenocortical mitochondria. 2. Amino acid residues of the apo-reductase were modified selectively, to identify the FAD-binding site of the reductase, with chemical reagents such as diethylpyrocarbonate, 5,5'-dithiobis(2-nitrobenzoate), tetranitromethane, pyridoxal 5'-phosphate, p-nitrophenylglyoxal, diisopropylfluorophosphate and N-bromosuccinimide. The binding of FAD to the apo-reductase was measured as quenching of the fluorescence of FAD caused by the binding between apo-reductase and FAD. The quenching was blocked when the apo-reductase was modified with diethylpyrocarbonate and restored on the addition of hydroxylamine. 3. The blocking of the quenching occurred in a competitive manner as to FAD in the presence of diethylpyrocarbonate. However, when the apo-reductase was modified with 5,5'-dithiobis(2-nitrobenzoate), the blocking of the quenching occurred in a non-competitive manner. 4. These results suggested that a histidyl residue of the apo-reductase is essential for the binding of FAD to the reductase. This was confirmed by amino acid sequencing of the modified apo-reductase.     

Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).     

Dihydroorotase from Escherichia coli. Sulfhydryl group-metal ion interactions

We have obtained 53 mg of 99% pure dihydroorotase from 10.9 g of frozen Escherichia coli pyrC plasmid-containing E. coli cells using a 4-step 16-fold purification procedure, a yield of 60%. We characterize the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (a dimer of subunit molecular weight 38,300 +/- 2,900), high performance liquid chromatography gel sieving, amino acid analysis, amino terminus determination (blocked), and specific activity. The isolated enzyme contains 1 tightly bound essential zinc atom/subunit, and readily but loosely binds 2 additional Zn(II) or Co(II) ions/subunit which modulate catalytic activity; treatment of crude extracts with weak chelators suggests that the enzyme contains 3 zinc atoms/subunit in vivo. Two of the 6 thiol groups/subunit react rapidly with 5,5'-dithiobis(2-nitrobenzoate) when 1 Zn/subunit enzyme is used, but slowly when 3 Zn/subunit enzyme is used. The 2 weakly bound Zn(II) ions/subunit protect against the reversible air oxidation which lowers the specific activity of the enzyme and renders it unreactive with 5,5'-dithiobis(2-nitrobenzoate). The dilution activation observed in the presence of substrate, the dilution inactivation observed in the absence of substrate, and the transient activation by the metal chelator oxalate are interpreted as evidence for an unstable, hyperactive monomer.     

Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans.

The enzyme 4-hydroxyphenylacetate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating) (EC 1.14.13 ...; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when 4-hydroxyphenylacetate (4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of 4-HPA. A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable. Flavin adenine dinucleotide and Mg2+ ions were required for full activity. 4-HPA 1-hydrocylase was inhibited by KCl, which was uncompetitive with 4-HPA. Values of Ki determined for inhibitors competitive with 4-HPA were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid. Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM 4-HPA, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH). The same maximum velocity was given by NADH and NADPH. A chemical synthesis is described for 2-deutero-4-hydroxyphenylacetic acid. This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed. Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of 4-HPA that possessed a hydroxyl group substituent at C-4 of the benze nucleus. A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid. Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity. This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring.     

Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH(2))-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH(2) in vitro, HpaB was able to use FADH(2) and O(2) for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH(2) for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH(2)-utilizing monooxygenase. FADH(2) generated by Fre was rapidly oxidized by O(2) to form H(2)O(2) in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH(2) and transitorily protected it from rapid autoxidation by O(2). When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O(2) for FADH(2) utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH(2) produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH(2) was autoxidized by O(2), causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH(2)-utilizing monooxygenase that uses FADH(2) as a substrate rather than as a cofactor.     

Partial purification and properties of a cyclic 3',5'-AMP-independent protein kinase from rat liver.

1. A cyclic 3',5'-AMP-independent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from rat liver cytosol was partially purified and characterized. Purification by (NH4)2SO4 precipitation, DEAE-cellulose, Bio Gel A-0.5 m and cellulose phosphate chromatography increased the specific activity about 700-fold. 2. An endogenous protein substrate was closely associated with the protein kinase and was not separable from this enzyme up to the cellulose phosphate stage. After phosphorylation, chromatography with Bio Gel A-0.5 m partially separated this endogenous phosphoprotein from the enzyme activity; this dissociation had no apparent effect on kinase activity with casein or phosvitin as substrates, or on the apparent molecular weight of the enzyme (approx. 158,000). 3. This protein kinase with casein, phosvitin, or the endogenous substrate was totally insensitive to the thiol reagents, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetamide, and N-ethylmaleimide. The enzyme was also unaffected by cyclic 3',5'-AMP, heat-stable protein kinase inhibitor, and the regulatory subunit of a cyclic 3',5'-AMP-dependent protein kinase.     

Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH₂)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH₂ in vitro, HpaB was able to use FADH₂ and O₂ for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH₂ for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH₂-utilizing monooxygenase. FADH₂ generated by Fre was rapidly oxidized by O₂ to form H₂O₂ in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH₂ and transitorily protected it from rapid autoxidation by O₂. When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O₂ for FADH₂ utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH₂ produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH₂ was autoxidized by O₂, causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH₂-utilizing monooxygenase that uses FADH₂ as a substrate rather than as a cofactor.     

Sulfhydryl groups of rabbit liver arylsulfatase A

Rabbit liver arylsulfatase A (aryl-sulfate sulfhydrolase, EC 3.1.6.1) monomers of 130 kDa contain two free sulfhydryl groups as determined by spectrophotometric titration using 5,5'-dithiobis(2-nitrobenzoate) and by labeling with the fluorescent probe 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Fluorescence quenching data indicate that the reactive sulfhydryl is present in proximity to one or more tryptophan residues. Chemical modification of the sulfhydryl groups does not alter the distinctive pH-dependent aggregation property of the arylsulfatase A. The free sulfhydryls of the enzyme react with numerous sulfhydryl reagents. Based on the reactions of iodoacetic acid, methyl methanethiosulfonate, 5,5'-dithiobis(2-nitrobenzoate) and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid with the sulfhydryl groups of arylsulfatase A, it is concluded that free sulfhydryls are not essential for the enzyme activity. In contrast, the observed inactivation of the enzyme by p-hydroxymercuribenzoate or p-hydroxymercuriphenylsulfonate is probably due to a modification of a histidine residue, consistent with previous reports that histidine is near the active site of arylsulfatase A. p-Hydroxymercuribenzoate and p-hydroxymercuriphenylsulfonate are able to react both with cysteine and with histidine residues of the protein molecule.     

Benzoyl-coenzyme A:glycine N-acyltransferase and phenylacetyl-coenzyme A:glycine N-acyltransferase from bovine liver mitochondria. Purification and characterization.

Two closely related acyl-CoA:amino acid N-acyl-transferases were purified to near-homogeneity from preparations of bovine liver mitochondria. Each enzyme consisted of a single polypeptide chain with a molecular weight near 33,000. One transferase was specific for benzoyl-CoA, salicyl-CoA, and certain short straight and branched chain fatty acyl-CoA esters as substrates while the other enzyme specifically used either phenylacetyl-CoA or indoleacetyl-CoA. Acyl-CoA substrates for one transferase inhibited the other. Glycine was the preferred acyl acceptor for both enzymes but either L-asparagine or L-glutamine also served. Peptide products for each transferase were identified by mass spectrometry. Enzymatic cleavage of acyl-CoA was stoichiometric with release of thiol and formation of peptide product. Apparent Km values were low for the preferred acyl-CoA substrates relative to the amino acid acceptors (10(-5) M range compared to greater than 10(-3) M). Both enzymes were inhibited by high nonphysiological concentrations of certain divalent cations (Mg2+, Ni2+, and Zn2+). In contrast to benzoyltransferase, phenylacetyltransferase was sensitive to inhibition by either 10(-4) M 5,5'-dithiobis(2-nitrobenzoate) or 10(-5) M p-chloromercuribenzoate; 10(-4) M phenylacetyl-CoA partially protected phenylacetyltransferase against 5,5'-dithiobis(2-nitrobenzoate) inactivation but 10(-1) M glycine did not. For activity, phenylacetyltransferase required addition of certain monovalent cations (K+, Rb+, Na+, Li+, Cs+, or (NH4)+) to the assay system but benzoyltransferase did not. Preliminary kinetic studies of both transferases were consistent with a sequential reaction mechanism in which the acyl-CoA substrate adds to the enzyme first, glycine adds before CoA leaves, and the peptide product dissociates last.     

Microbial degradation of hydrocarbons. Catabolism of 1-phenylalkanes by Nocardia salmonicolor

1. Nocardia salmonicolor grew on a variety of alkanes, 1-phenylalkanes and 1-cyclo-hexylalkanes as sole carbon and energy sources. 2. Growth on 1-phenyldodecane in batch culture was diauxic. Isocitrate lyase activity was induced during lag phase, reaching a maximum activity in the first growth phase, during which both the aromatic ring and the side chain were degraded. However, 4-phenylbutyrate, 4-phenylbut-3-enoate, 4-phenylbut-2-enoate, 3-phenylpropionate, cinnamate and phenylacetate accumulated in the growth medium. These compounds disappeared at the onset of diauxic lag and four hydroxylated compounds accumulated; one was 4-(o-hydroxyphenyl)but-3-enoate and another was identified as 4-(o-hydroxyphenyl)butyrate. These compounds were utilized during the second growth phase. 3. Washed 1-phenyldodecane-grown cells oxidized acetate, cinnamate, 3,4-dihydroxyphenylacetate, homogentisate, o-, m- and p-hydroxyphenylacetate, phenylacetate, and 4-phenylbutyrate rapidly without lag. 4. Extracts of such cells rapidly oxidized homogentisate,3,4-dihydroxyphenylacetate, catechol and protocatechuate. 5. The organism grew readily on 4-phenylbutyrate, phenylacetate, o-hydroxyphenylacetate, homogentisate and 3,4-dihydroxyphenylacetate as sole carbon energy sources, but growth was slow on cinnamate and 4-phenylbut-3-enoate. 6. When cinnamate and phenylacetate were sole carbon sources for growth, phenylacetate and o-hydroxyphenylacetate respectively were detected in culture supernatants. 4-Phenylbut-3-enoate and 4-phenylbutyrate both yielded a mixture of cinnamate and phenylacetate. 7. It is proposed that 1-phenyldodecane is catabolized by ω-oxidation of the terminal methyl group, side-chain β-oxidation to 4-phenylbutyrate, both β- and α-oxidation to phenylacetic acid, hydroxylation to homogentisate via o-hydroxyphenylacetate and ring cleavage to maleylacetoacetate. Catabolism via 3,4-dihydroxyphenylacetate may also occur. 8. Growth on 1-phenylnonane was also diauxic and cinnamic acid, phenylpropionic acid, benzoic acid and hydroxyphenylpentanoic acid accumulated in the medium. Respirometric data and ring-cleavage enzyme activities showed similar patterns to those obtained after growth on 1-phenyldodecane. The results suggest that the main catabolic routes for 1-phenyldodecane and 1-phenylnonane may converge at cinnamate. 9. Possible reasons for diauxie are discussed.     

Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.     

Glycylglycyl-L-cysteine as a substrate for renal sulfhydryl oxidase (glutathione oxidase)

Covalent chromatographically isolated bovine kidney sulfhydryl oxidase was found to catalyze the oxidation of cysteine and cysteine-containing substrates as determined by assaying with 5,5'-dithiobis(2-nitrobenzoate). Monitoring the time-course of substrate disappearance and product formation by means of high-pressure liquid chromatography revealed that such partially purified renal sulfhydryl oxidase preparations catalyze the direct oxidation of glycylglycyl-L-cysteine to its disulfide form with no other detectable metabolic products. Accordingly, Gly-Gly-Cys appears to be better suited for routine assays of sulfhydryl oxidase activity than is the traditionally employed substrate, glutathione, whose oxidation can be initiated by gamma-glutamyltransferase-catalyzed cleavage of the gamma-peptide bond, leading to falsely 'positive' assays in the absence of sulfhydryl oxidase per se.     

Catabolism of 3- and 4-hydroxyphenylacetate by the 3,4-dihydroxyphenylacetate pathway in Escherichia coli.

Various strains of Escherichia coli (but not strain K-12) were found to grow on 3-hydroxyphenylacetate and 4-hydroxyphenylacetate. Both compounds were catabolized by the same pathway, with 3,4-dihydroxyphenylacetate as a substrate for fission of the benzene nucleus, and with pyruvate and succinate as products. All the necessay enzymes were demonstrated in cell extracts prepared from induced cells but were essentially absent from uninduced cells. Mutants unable to grow on 3- and 4-hydroxyphenylactetate were defective in particular enzymes of the pathway. The characteristics of certain mutants indicated that either uptake or hydroxylation of 3- and 4-hydroxyphenylacetate may involve a common protein component. E. coli also grew on 3,4-hydroxyphenylacetate, with induction of the enzyme necessary for its degradation but not those for the uptake-hydroxylation of 3- and 4-hydroxyphenylacetate.     

3-Hydroxybenzoate 6-hydroxylase from Pseudomonas aeruginosa

An inducible 3-hydroxybenzoate 6-hydroxylase has been purified to homogeneity from $\text{Pseudomonas aeruginosa}$. It contains FAD as a prosthetic group. 3-Hydroxybenzoate is quantitatively hydroxylated to give gentisate with equimolar consumptions of NADH and O₂. NADPH will substitute as an electron donor, and several aromatic analogues of 3-hydroxybenzoate stimulate reduced nucleotide oxidation by the enzyme with formation of both hydrogen peroxide and hydroxylated products. Of various analogues of 3-hydroxybenzoate, those substituted in 2,4,5 and 6-positions are competent substrates; partial uncoupling of electron flow from hydroxylation with concomitant formation of hydrogen peroxide and “gentisates” occurs. The “natural” product of the reaction, gentisate, is an effector in that it stimulates NADH oxidation with the formation of hydrogen peroxide. 3-hydroxybenzoate 6-hydroxylase thus resembles other flavoprotein hydroxylases in the general regulatory properties dictated by their aromatic substrates, pseudosubstrates or effectors.     

Studies on regulatory functions of malic enzymes. IV. Effects of sulfhydryl group modification on the catalytic function of NAD-linked malic enzyme from Escherichia coli.

Reactions catalyzed by NAD-linked malic enzyme from Escherichia coli were investigated. In addition to L-malate oxidative decarboxylase activity (Activity 1) and oxaloacetate decarboxylase activity (Activity 2), the enzyme exhibited oxaloacetate reductase activity (Activity 3) and pyruvate reductase activity (Activity 4). Optimum pH's for Activities 3 and 4 were 4.0 and 5.0, and their specific activities were 1.7 and 0.07, respectively. Upon reaction with N-ethylmaleimide (NEM), Activity 1 decreased following pseudo-first order kinetics. Activity 2 decreased in parallel with Activity 1, while Activities 3 and 4 were about ten-fold enhanced by NEM modification. Modification of one or two sulfhydryl groups per enzyme subunit caused an alteration of the activities. Tartronate, a substrate analog, NAD+, and Mn2+ protected the enzyme against the modification. The Km values for the substrates and coenzymes were not significantly affected by NEM modification. Similarly, other sulfhydryl reagents such as p-hydroxymercuribenzoate (PMB), 5,5'-dithiobis(2-nitrobenzoate) (DTNB), and iodoacetate inhibited the decarboxylase activities and activated the reductase activities to various extents. Modification of the enzyme with PMB or DTNB was reversed by the addition of a sulfhydryl compound such as dithiothreitol or 2-mercaptoethanol. Based on the above results, the mechanism of the alteration of enzyme activities by sulfhydryl group modification is discussed.     

Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A

A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A-5,5'-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from epsilon(max) = 1.36 x 10(4) at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 10(-4) M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.     

Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: induction, purification, and characterization

A single strain of Pseudomonas cepacia cells was differentially induced to synthesize salicylate hydroxylase, 3-hydroxybenzoate 6-hydroxylase, or 4-hydroxybenzoate 3-hydroxylase. A procedure was developed for the purification of 3-hydroxybenzoate 6-hydroxylase to apparent homogeneity. The purified hydroxylase appears to be a monomer with a molecular weight of about 44,000 and exhibits optimal activity near pH 8. The hydroxylase contains one FAD per enzyme molecule and utilizes NADH and NADPH with similar efficiencies. The reaction stoichiometry for this enzyme has been determined. In comparison with other aromatic flavohydroxylases, this enzyme is unique in inserting a new hydroxyl group to the substrate at a position para to an existing one.     

Tritrichomonas foetus: purification and characterization of hydrogenosomal ATP:AMP phosphotransferase (adenylate kinase)

The hydrogenosomal enzyme ATP:AMP phosphotransferase (adenylate kinase) (EC 2.7.4.3) was purified to apparent homogeneity from the bovine parasite Tritrichomonas foetus. A fraction enriched for hydrogenosomes was obtained from cell homogenates which had been subjected to differential and isopycnic centrifugation. Adenylate kinase was solubilized in 50 mM Tris-HCl, pH 7.3, containing 0.8% Triton X-100, and purified by sequential Affi-Gel blue affinity chromatography and high-performance liquid chromatography gel filtration. The purified enzyme, a monomer of Mr 29,000, exhibited Km values of 100, 195, and 83 microM for ADP, ATP, and AMP, respectively. Substituting other mono-, di-, and trinucleotides for AMP, ADP, and ATP gave less than half the maximal activity. Full enzyme activity requires Mg2+, but Mn2+ and Co2+ yield half maximal activity. The enzyme has a broad optimal pH range between pH 6 and 9. The enzyme was competitively inhibited by P1,P5-di(adenosine-5')pentaphosphate, a specific adenylate kinase inhibitor: the Ki was 150 nM. The enzyme was also inhibited with 5,5'-dithiobis(2-nitrobenzoic acid), and this inhibition could be reversed by the addition of 2 mM dithiothreitol. T. foetus adenylate kinase has similar catalytic and physical properties to that of the biologically closely related human parasite Trichomonas vaginalis.     

Isolation and properties of N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase from Escherichia coli pABN11

The enzyme N epsilon-hydroxylysine acetylase has been isolated from Escherichia coli 294 carrying recombinant plasmid ABN11. Activity of the enzyme was followed by measurement of the rate of appearance of 2-nitro-5-thiobenzoate, the product of cleavage of 5,5'-dithiobis(2-nitrobenzoate) by free coenzyme A released from its acetyl derivative. The enzyme bound firmly to Reactive Blue 2-Sepharose CL-6B and was eluated with 1.5 M KCl. The protein gave a single band, corresponding to a Mr of 33,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In contrast, gel filtration of the native enzyme gave a Mr of 150,000-200,000. A sequence analysis of the DNA at the junction of the first and second genes in the aerobactin operon, considered in conjunction with the N-terminal amino acid sequence of the isolated protein, enabled the conclusion that the acetylase is specified by the second gene in the complex. The enzyme transfers the acetyl moiety from acetyl coenzyme A to a variety of hydroxylamines, with N epsilon-hydroxylysine as the preferred substrate. In agreement with the results found by affinity chromatography, Coomassie Blue was observed to act as a potent inhibitor.