Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase

The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. Ascorbate is virtually a specific requirement for these enzymes, but previous studies have demonstrated that it is not consumed during most catalytic cycles. Prolyl 4-hydroxylase and lysyl hydroxylase are known also to catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the peptide substrate. It is shown here that, unlike the complete hydroxylation reaction, the uncoupled decarboxylation reaction involves stoichiometric ascorbate consumption. This stoichiometric ascorbate consumption was also seen when the rate of the uncoupled prolyl 4-hydroxylase reaction was enhanced by the addition of poly(L-proline). Since collagen hydroxylases may catalyze occasional uncoupled reaction cycles even in the presence of the peptide substrates, the main function of ascorbate in these reactions in vivo is suggested to be that of reactivating the enzymes after such uncoupled cycles.     

Studies on the lysyl hydroxylase reaction. I. Initial velocity kinetics and related aspects

The kinetics of the lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) reaction were studied using enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double-reciprocal plots for all possible pairs involving Fe2+, alpha-ketoglutarate, O2 and the peptide substrate, whereas parallel lines were obtained for pairs comprising ascorbate and each of the other substrates. The pair composed of Fe2+ and alpha-ketoglutarate gave an asymmetrical initial veolcity pattern, indicating binding of these two reactants in this order, that of Fe2+ being at thermodynamic equilibrium. The initial velocity patterns are identical with those reported for prolyl 4-hydroxylase, and the apparent Km and Kd values calculated from these data are also very similar. The largest difference was fo-nd in Km and Kd for alpha-ketoglutarate, which were about 4 times the corresponding values for prolyl 4-hydroxylase. Ascorbate was found to be a quite specific requirement for lysyl hydroxylase, but the enzyme catalyzed its reaction for a short time at a high rate in the complete absence of this vitamin, suggesting that the reaction with ascorbate does not occur during each catalytic cycle. Lysyl hydroxylase catalyzed an uncoupled decarboxylation of alpha-ketoglutarate in the absence of the peptide substrate, the rate being about 4% of that observed in the presence of a saturating concentration of the peptide substrate. This uncoupled decarboxylation required the same cosubstrates as the complete reaction.     

Two-dimensional electrophoretic analysis of human erythrocyte cylindrin

In the absence of a peptidylproline substrate, the oxidative decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) is stoicheiometrically coupled to the oxidation of ascorbate. The Km and Kd for O2 in this partial reaction are 1.5 mM, this value being one order of magnitude higher than the Km and Kd for O2 in the complete reaction in the presence of (Pro-Pro-Gly)5, indicating that in this case O2 can become enzyme-bound predominantly after the interaction of the peptide substrate with the enzyme. The Km values for 2-oxoglutarate in the partial and the complete reactions are the same. In the absence of both a peptide substrate and ascorbate 2 mol CO2 per mol enzyme are produced in the first 1-1.5 min, during which the enzyme becomes inactivated and, as shown earlier (De Jong , L., Albracht , S.P.J. and Kemp, A. (1982) Biochim. Biophys. Acta 704, 326-332) enzyme-bound Fe2+ becomes oxidized to Fe3+. The results are consistent with a mechanism in which a Fe2+O complex is the O-transferring intermediate involved in peptidylproline hydroxylation.     

Characterization of alternate reductant binding and electron transfer in the dopamine beta-monooxygenase reaction

The steady-state limiting kinetic parameters Vmax, V/KDA, and V/KO2, together with deuterium isotope effects on these parameters, have been determined for the dopamine beta-monooxygenase (D beta M) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascorbate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of Vmax and V/KDA, and we conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/KO2 have been measured with [2,2-2H2]dopamine as substrate both in the presence and in the absence of added ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Markedly different ascorbate dependencies of the sequential alpha-ketoglutarate dioxygenase reactions catalyzed by an essentially homogeneous thymine 7-hydroxylase from Rhodotorula glutinis

The alpha-ketoglutarate dioxygenase, thymine 7-hydroxylase (EC 1.14.11.6), has been purified from cultures of Rhodotorula glutinis grown with thymine as a nitrogen source. The purification scheme developed yielded essentially homogeneous preparations of the 7-hydroxylase and also purified another alpha-ketoglutarate dioxygenase, pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3). The purity of the 7-hydroxylase was determined with analytical disc gel electrophoresis in which runs were varied with respect to pH, extent of cross-linking, and the presence of sodium dodecyl sulfate-mercaptoethanol. The 7-hydroxylase apparently exists as a monomer since its molecular weight was 42,700 when determined by molecular gel filtration chromatography and was 40,300 when determined by analytical disc gel electrophoresis under denaturing conditions. Gel filtration chromatography under nondenaturing conditions was used to show that the 2'-hydroxylase has a molecular weight of 64,600. The essentially homogeneous preparations of the 7-hydroxylase were shown to catalyze the thymine-, 5-hydroxymethyluracil-, and 5-formyluracil-dependent oxygenations that are coupled to the decarboxylation of alpha-ketoglutarate, as well as a putative uncoupled decarboxylation which is dependent on uracil. Furthermore, these enzyme preparations were used to show that ATP stimulated the 7-hydroxylase reaction in the absence of ascorbate. Even though it is attractive to consider the four pyrimidine-dependent reactions as being catalyzed by the same active site, they were shown to differ markedly in their dependencies on ascorbate or ATP. The effects of ascorbate and ATP on these reactions, and on the 2'-hydroxylase reaction, are discussed in terms of the possible roles of ascorbate and ATP.     

The role of ascorbate in protein folding

Ascorbate was linked to protein folding a long time ago. At the first level of this connection, it had been shown that ascorbate functions as an essential cofactor in the hydroxylation enzymes involved in collagen synthesis. Although the hydroxylation reactions catalyzed by the members of the prolyl 4-hydroxylase family are considered to be ascorbate dependent, the hydroxylation of proline alone does not need ascorbate. Prolyl 4-hydroxylases participate in two catalytic reactions: one in which proline residues are hydroxylated, while 2-oxoglutarate is decarboxylated and molecular oxygen is consumed. This reaction is ascorbate independent. However, in another reaction, prolyl 4-hydroxylases catalyze the decarboxylation of 2-oxoglutarate uncoupled from proline hydroxylation but still needing molecular oxygen. At this time, ferrous iron is oxidized and the protein is rendered catalytically inactive until reduced by ascorbate. At the second level of the connection, the oxidation and the oxidized form of ascorbate, dehydroascorbate, is involved in the formation of disulfide bonds of secretory proteins. The significance of the dehydroascorbate reductase activity of protein disulfide isomerase was debated because protein disulfide isomerase as a dehydroascorbate reductase was found to be too slow to be the major route for the reduction of dehydroascorbate (and formation of disulfides) in the endoplasmic reticulum lumen. However, very recently, low tissue ascorbate levels and a noncanonical scurvy were observed in endoplasmic reticulum thiol oxidase- and peroxiredoxin 4-compromised mice. This novel observation implies that ascorbate may be involved in oxidative protein folding and creates a link between the disulfide bond formation (oxidative protein folding) and hydroxylation.     

The function of ascorbate with respect to prolyl 4-hydroxylase activity

1. Incubation in the presence of 2-oxoglutarate and oxygen inactivates prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2), with a t 1/2 of 80 s at 37 degrees C. This inactivation is not affected by the presence or absence of the prolyl peptide substrate or added Fe(II). 2. This inactivation can be prevented by either ascorbate or dithiothreitol. It can be reversed by dithiothreitol but not by ascorbate. 3. Although the iron-containing form of prolyl 4-hydroxylase requires ascorbate for activity, ascorbate is not stoicheiometrically consumed in the reaction catalysed by the enzyme. Ascorbate cannot be replaced by alloxan, lactate, NADH plus phenazine methosulphate, dithiothreitol or L-cysteine. 4. Ascorbate has a double function with respect to prolyl 4-hydroxylase activity. On the one hand, it is required to initiate the reaction when the enzyme has become oxidized during isolation. On the other hand it is required for the protection against inactivation induced by 2-oxoglutarate and oxygen, presumably by preventing S-S bridge formation. The latter function may be of physiological importance.     

Uracil's uncoupling of the decarboxylation of alpha-ketoglutarate in the thymine 7-hydroxylase reaction of Neurospora crassa

Highly purified preparations of thymine 7-hydroxylase from Neurospora crassa catalyzed the decarboxylation of alpha-ketoglutarate but yielded no hydroxylated product when uracil was substituted for thymine in the standard incubation mixture. Although the uracil-dependent decarboxylation was much slower than the coupled reaction, both reactions were similar with respect to the requirement for molecular oxygen, the stoichiometric formation of succinate, and the stimulations effected by Fe2+, ascorbate, and catalase. That the same enzyme catalyzed both reactions was indicated by the parallel loss of the uracil- and thymine-dependent activities upon heat denaturation, their copurification, and the lower level of both activities in a mutant strain deficient in the 7-hydroxylase. These data are consonant with molecular oxygen initially attacking alpha-ketoglutarate in the thymine 7-hydroxylase reaction.     

Cloning of the alpha subunit of prolyl 4-hydroxylase from Drosophila and expression and characterization of the corresponding enzyme tetramer with some unique properties

Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, whereas the Caenorhabditis elegans enzyme is an alphabeta dimer, the beta subunit being identical to protein-disulfide isomerase (PDI). We report here that the processed Drosophila melanogaster alpha subunit is 516 amino acid residues in length and shows 34 and 35% sequence identities to the two types of human alpha subunit and 31% identity to the C. elegans alpha subunit. Its coexpression in insect cells with the Drosophila PDI polypeptide produced an active enzyme tetramer, and small amounts of a hybrid tetramer were also obtained upon coexpression with human PDI. Four of the five recently identified critical residues at the catalytic site were conserved, but a histidine that probably helps the binding of 2-oxoglutarate to the Fe2+ and its decarboxylation was replaced by arginine 490. The enzyme had a higher Km for 2-oxoglutarate, a lower reaction velocity, and a higher percentage of uncoupled decarboxylation than the human enzymes. The mutation R490H reduced the percentage of uncoupled decarboxylation, whereas R490S increased the Km for 2-oxoglutarate, reduced the reaction velocity, and increased the percentage of uncoupled decarboxylation. The recently identified peptide-binding domain showed a relatively low identity to those from other species, and the Km of the Drosophila enzyme for (Pro-Pro-Gly)10 was higher than that of any other animal prolyl 4-hydroxylase studied. A 1. 9-kilobase mRNA coding for this alpha subunit was present in Drosophila larvae.     

Determination of intra- and intermolecular tritium isotope effects in the reaction of thymine 7-hydroxylase

Different [7-3H]thymine preparations have been used to determine the inter- and intramolecular isotope effects of the 2-oxoglutarate-dependent thymine hydroxylation, catalyzed by thymine 7-hydroxylase (thymine, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.6). Specific activity ratios of products, viz., 3H2O and 5-hydroxymethyluracil, and remaining substrate to initial substrate have been determined. The influence on these ratios of intra- and intermolecular isotope effects at different degrees of tritium substitution has been analyzed. An intramolecular isotope effect with a kH/kT of about 6.5 has been found. No intermolecular isotope effect of TV/K could be detected when oxygen concentration was varied from 0.4 to 0.01 mM. This agrees with a mechanism in which 2-oxoglutarate is irreversibly changed before the bond-breaking in thymine takes place.     

Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in X-Pro-Gly sequences. The reaction requires Fe2+, 2-oxoglutarate, O2, and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. Ascorbate is not consumed during most catalytic cycles, but the enzyme also catalyzes decarboxylation of 2-oxoglutarate without subsequent hydroxylation, and ascorbate is required as a specific alternative oxygen acceptor in such uncoupled reaction cycles. A number of compounds inhibit prolyl 4-hydroxylase competitively with respect to some of its cosubstrates or the peptide substrate, and recently many suicide inactivators have also been described. Such inhibitors and inactivators are of considerable interest, because the prolyl 4-hydroxylase reaction would seem a particularly suitable target for chemical regulation of the excessive collagen formation found in patients with various fibrotic diseases. The active prolyl 4-hydroxylase is an alpha 2 beta 2 tetramer, consisting of two different types of inactive monomer and probably containing two catalytic sites per tetramer. The large catalytic site may be cooperatively built up of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit has been found to be identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and shows partial homology with a phosphoinositide-specific phospholipase C, thioredoxins, and the estrogen-binding domain of the estrogen receptor. The COOH-terminus of this beta subunit has the amino acid sequence Lys-Asp-Glu-Leu, which was recently suggested to be necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha subunit does not have this COOH-terminal sequence, and thus one function of the beta subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle.     

Partial identity of the 2-oxoglutarate and ascorbate binding sites of prolyl 4-hydroxylase

Various hydroxybenzenes, hydroxybenzoic acids, and related compounds resemble structurally both 2-oxoglutarate and ascorbate, two reactants needed in the reaction of prolyl 4-hydroxylase. These substances were found to inhibit prolyl 4-hydroxylase competitively with respect to both cosubstrates. Ortho-dihydroxy derivatives, which are capable of chelating the enzyme-bound iron, were the most effective inhibitors, with Ki values of about 5 microM. In contrast, pyridine 2-carboxylates, which have previously been reported to inhibit the enzyme competitively with respect to 2-oxoglutarate, were found to inhibit it uncompetitively with respect to ascorbate. In a separate set of experiments the side chain of the ascorbate molecule was shown to make no significant contribution to the binding of the reductant to the enzyme, as D(-)-isoascorbate and 5,6-O-isopropylidene ascorbate gave essentially the same Vmax and Km values as ascorbate. On the other hand, structural modifications of the ring atoms that abolished the chelating capacity destroyed both the cosubstrate and inhibitory activity, as in L-galactono gamma-lactone. The ascorbate binding site therefore appears to consist of two cis-positioned coordination sites of the enzyme-bound iron and is thus partially identical to the binding site of 2-oxoglutarate. This mode of interaction suggests that ascorbate reduces the enzyme-bound iron through an "inner-sphere" mechanism. The inhibitors studied appear to react at different phases of the catalytic cycle, determined by the oxidation state of the enzyme-bound iron atom.     

Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.     

Syncatalytic inactivation of prolyl 4-hydroxylase by synthetic peptides containing the unphysiologic amino acid 5-oxaproline

Peptides containing the unphysiological amino acid 5-oxaproline (Opr) in the sequence R1-Xaa-Opr-Gly-OR2 were found to inactivate prolyl 4-hydroxylase from chick and human origins. Of the substances investigated, compounds with aromatic substituents R1 and R2 were particularly effective when compared with those with an aliphatic group or without a C-terminal blocking group. Both affinity of the individual peptides for the enzyme and partition ratio contributed to the differences in efficiency. Benzylcarbonyl-Phe-Opr-Gly-benzyl ester was the most effective substance tested, its concentration giving 50% inactivation in 1 h being 0.8 microM. Inactivation was only observed in the presence of 2-oxoglutarate and Fe2+. The Opr peptides enhanced the decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase, the Vmax values obtained with the individual peptides being positively correlated with their inactivating efficiency. Inactivation was prevented by high concentrations of peptide substrate and ascorbate. Lineweaver-Burk kinetics experiments suggested noncompetitive inhibition with respect to peptide substrate and ascorbate. Lysyl hydroxylase was not affected by Opr peptides in concentrations of up to 1.5 mM in either the presence or absence of prolyl 4-hydroxylase. The results suggest that the oxaproline compounds are specific syncatalytic inactivators of prolyl 4-hydroxylase.     

Characterization of a low-relative-molecular-mass prolyl 4-hydroxylase from the green alga Chlamydomonas reinhardii.

Prolyl 4-hydroxylase was partially purified and characterized from the unicellular green alga, Chlamydomonas reinhardii. This enzyme differed from all the animal and plant prolyl 4-hydroxylases studied so far in that its Mr was only about 40,000 by gel filtration, being thus less than one-sixth of those determined for the vertebrate and higher-plant enzymes. The algal enzyme did not hydroxylate to any significant extent chick-embryo protocollagen or triple-helical (Pro-Pro-Gly)10, whereas a low hydroxylation rate was found with denatured (Pro-Pro-Gly)10. Poly(L-proline), which is an effective inhibitor of the vertebrate enzymes but acts as a substrate for some higher-plant enzymes, was a good substrate. In the absence of poly(L-proline) the enzyme catalysed an uncoupled decarboxylation of 2-oxoglutarate. Studies of the Km values for the co-substrates and cofactors and the specificity of the 2-oxoglutarate requirement, as well as inhibition studies with selected 2-oxoglutarate analogues, suggested that the catalytic site of the algal enzyme is similar to, but not identical with, those of the vertebrate enzymes. The existence of distinct similarities was further demonstrated by an inhibition of the algal enzyme activity with a monoclonal antibody to the beta-subunit of human prolyl 4-hydroxylase. The amount of prolyl 4-hydroxylase activity in the algal cells was not altered by signals which recognize the presence or absence of the cell wall, as determined in studies on experimental cell-wall regeneration and wall-less mutants.     

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.     

A kinetic study of thymine 7-hydroxylase from neurospora crassa.

The steady-state kinetics of thymine 7-hydroxylase (thymine, 2-oxoglutarate dioxygenase, EC 1.14.11.6) has been investigated. Initial velocity plots were all found to be linear and intersecting. Variation in concentration of two of the substrates, when the third substrate was at a constant high or low concentration, gave initial velocity plots that conform to an ordered sequential mechanism, where thymine is the second substrate to add. With 5-carboxyuracil, which is the end product in the sequential oxygenation of thymine, a competitive inhibition pattern was observed when 2-ketoglutarate was the variable substrate. When either thymine or oxygen was the variable substrate a noncompetitive inhibition pattern was obtained. When either 2-ketoglutarate or thymine was the variable substrate the inhibition patterns observed with bicarbonate were noncompetitive. With succinate noncompetitive inhibition patterns with hyperbolic intercept replots were obtained. These results are consistent with an ordered sequential kinetic mechanism, where 2-ketoglutarate is added first, followed by thymine and oxygen, and the products are released in the order: bicarbonate, succinate, and 5-hydroxymethyluracil. The order of the two last mentioned products, however, is changed in the presence of succinate.     

Thymine 7-hydroxylase and pyrimidine deoxyribonucleoside 2' -hydroxylase activities in Rhodotorula glutinis

Cell-free preparations from Rhodotorula glutinis catalyzed the conversion of deoxyribonucleosides to ribonucleosides in a pyrimidine deoxyribonucleoside 2' -hydroxylase reaction. The reaction occurred with only thymidine or deoxyuridine, of the common deoxyribonucleosides, without detachment of the deoxyribose moiety, at the nucleoside level. The same enzyme preparations catalyzed the conversion of thymine to 5-hydroxymethyluracil in a thymine 7-hydroxylase reaction. Requirements for molecular oxygen, alpha-ketoglutarate, Fe2+, and ascorbate indicated that the 2' -hydroxylase and 7-hydroxylase reactions are of the alpha-keto-acid dioxygenases class. The requirements for alpha-ketoglutarate and Fe2+ were very stringent. During the course of the 2' -hydroxylase and 7-hydroxylase reactions, alpha-ketoglutarate was decarboxylated to form succinate and CO2 so that the ratio of hydroxylated nucleoside or pyrimidine to CO2 was 1:1.5-Hydroxymethyluracil and 5-formyluracil also stimulated the decarboxylation of alpha-ketoglutarate and thus appeared to undergo 7-hydroxylase reactions.     

Use of alternate substrates to probe the order of substrate addition to dopamine beta-hydroxylase

In order to determine the order of substrate binding to dopamine beta-hydroxylase during catalysis, the effect of alternate substrates upon kinetic parameters was examined. The V/K value for ascorbate was unchanged when tyramine, phenylpropylamine, p-Cl-phenethylamine, p-CH3O-phenethylamine, or phenethylamine was the hydroxylated substrate. The V/K values for tyramine and oxygen were similarly unchanged when ferrocyanide was used as the reductant in place of ascorbate. In order to use ferrocyanide as reductant it was necessary to include copper to alleviate the substrate inhibition seen with this substrate. The pattern of substrate inhibition observed with ferrocyanide was consistent with a small amount of free cyanide present in the ferrocyanide. With ferrocyanide as reductant and [2,2-2H2]tyramine as substrate, there was a measurable isotope effect on the V/K value for oxygen, but none on the values of Vmax or V/K for tyramine. These results are consistent with a ping-pong mechanism in which tyramine binds to the enzyme after the release of oxidized ascorbate. Subsequently, oxygen binds to form a ternary complex.     

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.