An adenosine triphosphate-dependent carbamoylphosphate--3-hydroxymethylcephem O-carbamoyltransferase from Streptomyces clavuligerus.

Cell-free supernatants from cells of Streptomyces clavuligerus (N.R.R.L. 3585), which are actively synthesizing cephamycin C, transfer a carbamoyl group from carbamoylphosphate to a 3-hydroxymethylceph-3-em-4-carboxylic acid nucleus to form a 3-carbamoyloxymethylcephem. This reaction was stimulated by nucleoside triphosphates and by a mixture of Mn2+ and Mg2+ cations. The enzyme responsible was purified 40-fold by batch absorption onto DEAE-cellulose and hydroxyapatite. The purified O-carbamoyltransferase is most active at pH 6.8. It is stabilized by phosphate anions, but is inhibited by PPi anions, (NH4)2SO4 or NaCl. The enzyme is stimulated by ATP, but it is not known whether this nucleotide acts as an effector or as a substrate. Some activity is observed with dATP, but two other analogues of ATP, in which a methylene group replaced the oxygen atom between the alpha- and beta- or the beta- and gamma-phosphorus atoms, inhibit the action of ATP itself. The enzyme synthesizes a wide range of 3-carbamoyloxymethylcephems. The structure of some of these products, for example that of cefuroxime (3-carbamoyloxymethyl-7 beta-[2-(fur-2-yl)-2-syn-methoxyiminoacetamido]ceph-3-em-4-carboxylic acid), was confirmed by their proton-n.m.r. spectra.     

Purification of isopenicillin N synthetase.

Isopenicillin N synthetase was extracted from Cephalosporium acremonium and purified about 200-fold. The product showed one major protein band, coinciding with synthetase activity, when subjected to electrophoresis in polyacrylamide gel. An isopenicillin N synthetase from Penicillium chrysogenum was purified about 70-fold by similar procedures. The two enzymes resemble each other closely in their Mr, in their mobility on electrophoresis in polyacrylamide gel and in their requirement for Fe2+ and ascorbate for maximum activity. Preliminary experiments have shown that a similar isopenicillin N synthetase can be extracted from Streptomyces clavuligerus.     

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.     

Studies on the active site of deacetoxycephalosporin C synthase

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; alpha=beta=90 degrees, gamma=120 degrees (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.     

Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida

(2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2-oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200-fold by a six-step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74,000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2-oxoglutarate, Fe2+ and ascorbate. With 2-oxo[1-14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)-(2R,3R)-dihydrokaempferol. (2S)-Naringenin, but not the (2R)-enantiomer is a substrate of the hydroxylase. (2S)-Eriodictyol is converted to (2R,3R)-dihydroquercetin. In contrast, 5,7,3',4',5'-pentahydroxy-flavanone is not a substrate. Apparent Michaelis constants for (2S)-naringenin and 2-oxoglutarate were determined to be respectively 5.6 mumol X l-1 and 20 mumol X l-1 at pH 8.5. The Km for (2S)-eriodictyol is 12 mumol X l-1 at pH 8.0. Pyridine 2,4-dicarboxylate and 2,5-dicarboxylate are strong competitive inhibitors with respect to 2-oxoglutarate with Ki values of 1.2 mumol X l-1 and 40 mumol X l-1, respectively.     

Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus

A putatively rate-limiting synthase (expandase) of Streptomyces clavuligerus was stabilized in vitro and purified 46-fold from cell-free extracts; a major enriched protein with a Mr of 35,000 was further purified by electrophoretic elution. Based on a 22-residue amino-terminal sequence of the protein, the synthase gene of S. clavuligerus was cloned and expressed in Escherichia coli (Kovacevic, S., Weigel, B.J., Tobin, M.B., Ingolia, T.D., and Miller, J. R. (1989) J. Bacteriol. 171, 754-760). The synthase protein was detected mainly from granules of recombinant E. coli. The recombinant synthase was solubilized from the granules by urea, and for the first time a highly active synthase was purified to near homogeneity. The synthase was a monomer with a Mr of 34,600 and exhibited two isoelectric points of 6.1 and 5.3. Its catalytic activity required alpha-ketoglutarate, Fe2+, and O2, was stimulated by dithiothreitol or ascorbate but not by ATP, and was optimal at pH 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and at 36 degrees C. The Fe2+ requirement was specific, and at least one sulfhydryl group in the purified enzyme was apparently essential for the ring expansion. The Km values of the enzyme for penicillin N and alpha-ketoglutarate were 29 and 18 microM, respectively, and the Ka for Fe2+ was 8 microM. The recombinant synthase was indistinguishable from the native synthase of S. clavuligerus by those biochemical properties. In addition to the enzymic ring expansion of penicillin N to deacetoxycephalosporin C, the recombinant synthase catalyzed a novel hydroxylation of 3-exomethylenecephalosporin C to deacetylcephalosporin C.     

Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.     

Purification and properties of an ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2.

A novel ethylene-forming enzyme that catalyses the formation of ethylene from 2-oxoglutarate was purified from a cell-free extract of Pseudomonas syringae pv. phaseolicola PK2. It was purified about 2800-fold with an overall yield of 53% to a single band of protein after SDS-PAGE. The purified enzyme had a specific activity of 660 nmol ethylene min-1 (mg protein)-1. The molecular mass of the enzyme was approximately 36 kDa by gel filtration and 42 kDa by SDS-PAGE. The isoelectric point and optimum pH were 5.9 and ca. 7.0-7.5, respectively. There was no homology between the N-terminal amino acid sequence of the ethylene-forming enzyme of Ps. syringae pv. phaseolicola PK2 and the sequence of the ethylene-forming enzyme of the fungus Penicillium digitatum IFO 9372. However, the two enzymes have the following properties in common. The presence of 2-oxoglutarate, L-arginine, Fe2+ and oxygen is essential for the enzymic reaction. The enzymes are highly specific for 2-oxoglutarate as substrate and L-arginine as cofactor. EDTA, Tiron, DTNB [5,5'-dithio-bis(2-nitrobenzoate)] and hydrogen peroxide are all effective inhibitors.     

The conversion of cephalosporins to 7 alpha-methoxycephalosporins by cell-free extracts of Streptomyces clavuligerus.

In the presence of S-adenosylmethionine, 2-oxoglutarate, Fe2+ and a reducing agent, cell-free extracts of Streptomyces clavuligerus convert cephalosporin C and O-carbamoyldeacetylcephalosporin C into 7 alpha-methoxy derivatives. No synthesis of a 7 alpha-methoxy derivative of deacetylcephalosporin C was detected in the system used, and the 7 alpha-methoxy derivative of deacetoxycephalosporin C was produced only in relatively small amounts. It appears that the 7 alpha-methoxy group is introduced after the cephalosporin ring system has been formed and that its introduction may represent the final step in a biosynthetic pathway.     

Prevention of phosphate inhibition of cephalosporin synthetases by ferrous ion

Abstract Phosphate interference in the production of cephalosporins by Streptomyces clavuligerus had been associated with repression of expandase (desacetoxycephalosporin C synthetase) and inhibition of both expandase and cyclase (isopenicillin N synthetase). The present work shows that inhibition of enzyme action could be prevented by increasing the Fe²⁺ added to the cell-free reactions or to resting cells incubated with chloramphenicol. Since excess Fe²⁺ could not reverse phosphate interference of antibiotic synthesis in complete fermentations, it is clear that the major cause of the phosphate effect in fermentations is phosphate repression, rather than phosphate inhibition caused by Fe²⁺ deprivation.     

Purification and characterization of hyoscyamine 6 beta-hydroxylase from root cultures of Hyoscyamus niger L. Hydroxylase and epoxidase activities in the enzyme preparation

Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l-hyoscyamine to 6 beta-hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y. (1986) Plant Physiol. 81, 619-625] was purified 310-fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 41,000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8 l-Hyoscyamine and 2-oxoglutarate are required for the enzyme activity, with respective Km values of 35 microM and 43 microM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2-Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested. The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o-dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4-dicarboxylate and 3,4-dihydroxybenzoate are competitive inhibitors with respect to 2-oxoglutarate with the respective Ki values of 9 microM and 90 microM. Several alkaloids with structures similar to l-hyoscyamine were hydroxylated by the enzyme at the C-6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7-dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 microM). This epoxidation reaction required the same co-factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6 beta-hydroxylase.     

Desacetoxycephalosporin C synthetase: Importance of order of cofactor/ reactant addition

The ring expansion enzyme (desacetoxycephalosporin C synthetase) of cephalosporin C biosynthesis in Cephalosporium acremonium has been purified 40-fold, using gel filtration on LKB Ultrogel AcA54. Purity was about twice that previously attained. The purified enzyme showed an almost absolute requirement for α-ketoglutarate, and most other acids were inactive or only weakly active. The order of addition of the cofactors and reactants was found to exert a major effect on enzyme activity. The major negative effect was caused by α-ketoglutarate or penicillin N being added to the enzyme first. The most effective order of addition to the enzyme was found to be the simultaneous addition of Fe²⁺, ascorbate and ATP, followed by α-ketoglutarate and then penicillin N. The negative effect of the required reactant, α-ketoglutarate, when added too early, coincides with observations made about other α-ketoglutarate-dependent dioxygenases even though, in the case of the synthetase, one atom of oxygen does not end up in the product, desacetoxycephalosporin C.     

Isopenicillin N synthase and desacetoxycephalosporin C synthase activities during defined medium fermentations of Streptomyces clavuligerus: effect of oxygen and iron supplements

When the level of dissolved oxygen was increased to saturation in defined media fermentations of Streptomyces clavuligerus, the total duration of activity of the penicillin ring cyclization enzyme, isopenicillin N synthase (IPNS), was extended by at least 20 h; however, no increase in the stability of the ring expansion enzyme, desacetoxycephalosporin C synthase (DAOCS), was observed. Consequently, the conversion of the excreted intermediate penicillin N to cephamycin C was 15-20% less efficient at this high oxygen concentration. The increased dissolved oxygen level also led to the complete loss of IPNS and DAOCS activities for 4 h during the period of fastest growth, and the rate of specific cephamycin C production fell to zero. A several hundred fold increase in the level of iron in the defined media resulted in a sixfold improvement in the rate of specific cephamycin C production after 60 h fermentation. This increased rate appeared to be due to an elevation in the in vivo activities of a number of the cephamycin biosynthetic enzymes, particularly those catalysing later pathway steps.     

Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans

The deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamdurans was highly purified, as shown by SDS-PAGE and isoelectric focusing. The enzyme catalysed the oxidative ring expansion that converts penicillin N into DAOC. The enzyme was very unstable but could be partially stabilized in 25 mM-Tris/HCl, pH 9.0, in the presence of DTT (0.1 mM). The enzyme required 2-oxoglutarate, oxygen and Fe2+, but did not need ATP, ascorbic acid, Mg2+ or K+. The optimum temperature was between 25 and 30 degrees C. The DAOC synthase showed a high specificity for the penicillin substrate. Only penicillin N but not isopenicillin N, penicillin G or 6-aminopenicillanic acid served as substrates. 2-Oxoglutarate analogues were not used as substrates although 2-oxobutyrate and 3-oxoadipate inhibited the enzyme by 100% and 56% respectively. The enzyme was strongly inhibited by Cu2+, Co2+ and Zn2+. The apparent Km values for penicillin N, 2-oxoglutarate and Fe2+ were 52 microM, 3 microM and 71 microM respectively. The enzyme was a monomer with a molecular mass of 27,000 Da +/- 1,000.     

Expression of the transporter encoded by the cefT gene of Acremonium chrysogenum increases cephalosporin production in Penicillium chrysogenum

By introduction of the cefEF genes of Acremonium chrysogenum and the cmcH gene of Streptomyces clavuligerus, Penicillium chrysogenum can be reprogrammed to form adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA), a carbamoylated derivate of adipoyl-7-aminodeacetoxy-cephalosporanic acid. The cefT gene of A. chrysogenum encodes a cephalosporin C transporter that belongs to the Major Facilitator Superfamily. Introduction of cefT into an ad7-ACCCA-producing P. chrysogenum strain results in an almost 2-fold increase in cephalosporin production with a concomitant decrease in penicillin by-product formation. These data suggest that cephalosporin production by recombinant P. chrysogenum strains is limited by the ability of the fungus to secrete these compounds.     

Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation

Dithiothreitol (DTT) has been found to stimulate the desacetoxycephalosporin C synthetase (expandase) activity of a frozen crude extract of Streptomyces clavuligerus, even in the presence of an optimum concentration of ascorbate. Catalase, both native and heat-denatured, and bovine serum albumin (BSA) also stimulated the enzyme activity but were less active than DTT. Although fresh extracts were sporadically stimulated, frozen extracts or extracts inactivated by shaking at 37°C in air were consistently activated. It is felt that DTT functions as a reactivating, rather than an activaing, agent. Similar effects were observed with extracts of Cephalosporium acremonium.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity

The biosynthesis of cephalosporins involving a thiozolidine ring expansion is catalyzed by deacetoxycephalosporin C synthase (DAOCS). In this study, three DAOCS isozymes were cloned and expressed as active enzymes together with Streptomyces jumonjinensis DAOCS that was newly isolated and partially characterized. The enzymes showed excellent substrate conversion for penicillin G, phenethicillin, ampicillin and carbenicillin, but they were less effective in the ring expansion of penicillin V, amoxicillin and metampicillin. Streptomyces clavuligerus DAOCS was the most active among the recombinant enzymes. The results also showed that truncation of 20 amino acids at the C-terminus of the Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase polypeptide did not affect penicillin ring expansion.     

Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.     

Assay for 2-oxoglutarate decarboxylating enzymes based on the determination of [1-14C]succinate: application to prolyl 4-hydroxylase

A method for the assay of 2-oxoglutarate decarboxylating enzymes based on determination of the reaction product [1-14C]succinate after precipitation of remaining 2-oxo[5-14C]glutarate with 2,4-dinitrophenyl hydrazine is reported. It is particularly useful for the study of the 2-oxoglutarate-coupled dioxygenase prolyl 4-hydroxylase (EC 1.14.11.2); it is superior to previously described assay methods of this enzyme with respect to simplicity of the procedure, speed, cost, and radiochemical safety. The results are highly reproducible, the standard deviation of repeated measurements being about 2% of the mean. The commercially available 2-oxo[5-14C]glutarate used in this study contained approximately 3% of radioactivity coeluting with succinate in HPLC and 1.5% of an unidentified radioactive compound as impurities, which contributed to the background.