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B Entsch

Publications and source records attributed to B Entsch.

29 records · Page 2Linked to original sources
PubMed1987

para-Hydroxybenzoate hydroxylase containing 6-hydroxy-FAD is an effective enzyme with modified reaction mechanisms.

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens, was replaced by 6-hydroxy-FAD (an extra hydroxyl group on the carbon at position 6 of the isoalloxazine ring of FAD). The catalytic cycle of this modified enzyme was analyzed and compared to the function of native (FAD) enzyme. Transient state kinetic analyses of the multiple changes in the chemical state of the flavin were the principal methods used to probe the mechanism. Four known substrates of the native enzyme were used to probe the reaction. With the natural substrate, p-hydroxybenzoate, the 6-hydroxy-FAD enzyme activity was 12-15% of native enzyme, due to a slower release of product from the enzyme, and less than one product molecule was formed per NADPH oxidized, due to an increased rate of nonproductive decomposition of the transient peroxyflavin essential to the catalytic pathway. More extensive changes in mechanism were observed with the substrates, 2,4-dihydroxybenzoate and p-aminobenzoate. The results suggest that, during catalysis, when the reduced state of FAD is ready for oxygen reaction, the substrate is located below and close to the C-4a/N-5 edge of the isoalloxazine ring. The nature of the high extinction, transient state of flavin, formed upon transfer of oxygen to substrate is discussed. It is not a flavin cation, and is unlikely to be an oxygen-substituted analogue of N-3/C-4 dihydroflavin.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1983

The purification and identification of flavin nucleotides by high-performance liquid chromatography.

Many preparations of flavin nucleotides contain nucleotide isomers of the natural compounds which are difficult to remove or separate. The method of dynamic complex-exchange (or paired-ion) chromatography has been used with high-performance liquid chromatography to achieve resolution and purification of isomers. A solution of nucleotide in water was chromatographed isocratically on a C18-substituted silica column with a mobile phase of methanol, water, and tetrabutylammonium phosphate at neutral pH. Commercial preparations of FMN and FAD contained multiple components. The purified isomers were subjected to ion-exchange chromatography directly on a quaternary nitrogen-substituted silica column to remove methanol and tetrabutylammonium cation, and thus obtain pure nucleotide in aqueous buffer suitable for use with proteins. With analytical equipment, a milligram of pure FMN or FAD was produced in 1 day. The same procedure was useful for the rapid identification and quantitation of flavin nucleotides in proteins. After exposure of a protein solution to heat treatment, the supernatant was subjected to dynamic complex-exchange chromatography, as described above.

Chromatography, High Pressure Liquid↗
PubMed1980

Fluoride elimination from substrates in hydroxylation reactions catalyzed by p-hydroxybenzoate hydroxylase.

Several fluorinated derivatives of p-hydroxybenzoate were synthesized and examined as substrates in the reaction catalyzed by p-hydroxybenzoate hydroxylase. All the derivatives tested served as substrates, undergoing tightly coupled hydroxylation by molecular oxygen. Hydroxylation of the difluoro and tetrafluoro derivatives liberated stoichiometric amounts of fluoride. Little or no fluoride was released with monofluoro substrates. The defluorination caused higher consumption of NADPH with an overall NADPH to oxygen ratio of 2, in contrast to the ratio of 1 with the physiological substrate and with the monofluoro derivatives. Evidence was obtained strongly suggestive of a quinonoid species as the primary product formed upon oxygenative defluorination. The additional equivalent of NADPH consumed upon fluoride elimination is presumably used in a nonenzymatic reaction with the quinonoid intermediate, resulting in the observed dihydroxy product. Stopped flow studies of the reductive and oxidative half-reactions with tetrafluoro-p hydroxybenzoate substrate were examined. The oxygen half-reaction was analogous to that with p-hydroxybenzoate involving two transient oxygenated flavin intermediates. The decay of the first intermediate, a C(4a)-peroxyflavin, results in rupture of the oxygen-oxygen bond and is rate-determining in overall catalysis. This is in contrast to the reaction with the normal substrate, presumably due to a deactivating effect of the fluorine substituents. The above results are consistent with an oxenoid mechanism of oxygen attack.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1980

Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD.

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1979

Preparation and characterization, using high-performance liquid chromatography, of an enzyme forming glucosides of cytokinins.

Cytokinins can occur naturally as glycosides with beta-D-glucose as the sugar substituent. From radish (Raphanus sativus) cotyledons, an enzyme has been partly purified which synthesizes the 7-glucopyranoside of zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine], a compound known to occur in this species. High-performance reverse-phase liquid chromatography was uniquely useful as the analytical procedure for quantitative study of the minute amounts of enzyme available. The enzyme uses UDPglucose as the source of the sugar residue. A large number of derivatives of purine are glucosylated, but adenine derivatives with an alkyl side chain at least three carbon atoms in length at position N6 are preferentially glucosylated. This corresponds to the structural features required for high cytokinin activity. The 7-glucoside of zeatin is known to be very weakly active in cytokinin bioassays. Hence, this enzyme, and others catalyzing the same reaction, have a role in the regulation of cytokinin activity.

Cytokinins↗
PubMed1977

On the structure of flavin-oxygen intermediates involved in enzymatic reactions.

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421--505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C(4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the subsituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1976

Catalytic mechanism of p-hydroxybenzoate hydroxylase with p-mercaptobenzoate as substrate.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens catalyzes in vivo the hydroxylation of p-hydroxybenzoate by molecular oxygen to form 3,4-dihydroxybenzoate. p-Mercaptobenzoate is also a substrate of the enzyme, but instead of being converted to the expected product, 3-hydroxy-4-mercaptobenzoate, the disulfide, 4,4'-dithiobisbenzoate, is formed. To find what mechanistic information this unusual reaction provided, steady state kinetic analyses, combined with rapid reaction studies of the changes in the enzyme-bound FAD, were carried out with the separate half-reactions involved in catalysis. Most of the kinetic measurements were made with a stopped-flow spectrophotometer designed for working anaerobically and connected on line to a minicomputer. Initial rate studies, upon varying systematically the concentrations of p-mercaptobenzoate, NADPH, and oxygen showed that the enzyme interacted with the substrates in the same manner as it does with p-hydroxybenzoate in place of the mercaptan. That is, a ternary complex is formed between enzyme, mercaptobenzoate, and NDAPH, followed by reaction and release of NADP+. Then a second ternary complex is formed between enzyme, mercaptobenzoate, and oxygen followed by reaction, liberation of product, and return to the resting state of the enzyme. Rapid reaction studies showed that the first half-reaction was analagous to that with the natural substrate. The enzyme-flavin is reduced to the 1,5-dihydroflavin by NADPH, and the rate of reaction is dramatically enhanced in the presence of mercaptobenzoate. The rate enhancement with this enzyme correlates well with the presence of a dianion form of the substrate on the enzyme. Examination of the second half-reaction showed that the reduced flavin on the enzyme formed transient intermediates upon reaction with oxygen, which were analogous to the intermediates in reactions where the enzyme forms an hydroxylated product. The oxidation of p-mercaptobenzoate by H2O2 in free solution resulted in the same disulfide as formed in the enzymatic reaction, only orders of magnitude slower. A sulfenic acid was probably the initial oxidation product from p-mercaptobenzoate, and this reacted very fast, and nonenzymatically, with mercaptobenzoate to form the disulfide and H20. The significance of the enzyme reaction with oxygen when complexed with p-mercaptobenzoate is discussed in relation to the mechanism of hydroxylation.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1976

Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase.

Para-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is one of a group of flavoproteins which insert molecular oxygen into aromatic rings to form phenols. To determine the mechanism of oxygen insertion by this enzyme, an extensive study was made of the reaction with O2 of reduced enzyme in complex with various aromatic molecules. Reactions were studied by following absorbance changes with time with a stopped-flow spectrophotometer. Analysis of multiphasic reactions led to the detection of a minimum of three transient intermediates with characteristic absorption spectra involved in the process of hydroxylation. The initial interaction of oxygen with the reduced enzyme characteristically produces a derivative of FAD (maximum absorbance 380 to 390 nm) which is probably C(4a) peroxyflavin. Depending on the aromatic compound bound to the enzyme, this intermediate decays either to oxidized, enzyme-bound flavin and H2O2 or transfers an atom of oxygen to the aromatic compound. The process of oxygen transfer forms a derivative of FAD of unknown structure (maximum absorbance 390 to 420 nm), which subsequently decays to the third intermediate observed (maximum absorbance 380 to 385 nm), which is probably C(4a) hydroxyflavin. The decay of this last intermediate results in the formation of oxidized enzyme, and the liberation of hydroxylated product and H2O. In an extension of substrate specificity studies it was found that p-aminobenzoate is a substrate and 5-hydroxypicolinate is an effector for p-hydroxybenzoate hydroxylase. The binding of aromatic compounds to the reduced enzyme was observed by following shifts in the absorption spectrum of enzyme bound FADH2, permitting the determination of dissociation constants and kinetics of binding.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1972

Nicotinamide adenine dinucleotide phosphate photoreduction from water by agranal chloroplasts isolated from bundle sheath cells of maize.

Photoreduction of NADP from water in agranal chloroplasts isolated from the leaf bundle sheath cells of Zea mays (var. DS 606A) or Sorghum bicolor (var. Texas 610) was dependent upon addition of plastocyanin as well as ferredoxin. Activity was further increased by the addition of ferredoxin NADP-reductase. Saturation for plastocyanin was reached at about 6 micromolar. In contrast, grana-containing chloroplasts isolated from leaf mesophyll cells of these plants or from pea (Pisum sativum L.) leaves did not require either plastocyanin or ferredoxin NADP-reductase for NADP photoreduction from water, although with some preparations plastocyanin stimulated the activity.Photosystem I activity, which was low in washed preparations of bundle sheath chloroplasts, was also stimulated by plastocyanin. The effect of plastocyanin on photosystem I activity in the grana-containing chloroplasts was similar to that on NADP photoreduction from water.In the presence of plastocyanin, the rates of NADP photoreduction from water were about the same in the agranal and granal chloroplasts, but photosystem I activity was considerably higher in bundle sheath chloroplasts. In these chloroplasts photosystem II appeared to limit the rate of NADP photoreduction.The results indicated that the agranal bundle sheath chloroplasts reduced plastocyanin via photosystem II and oxidized it via photosystem I. Both types of maize chloroplast photoreduced oxidized plastocyanin, but in the presence of methyl viologen, reduced plastocyanin was photo-oxidized only by the bundle sheath chloroplasts.

Journal Article↗