A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.     

Isolation and partial characterization of a vitamin K-dependent carboxylase from bovine aortae.

Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu ('FLEEL'), decarboxylated osteocalcin, decarboxylated fragment 13-29 from descarboxyprothrombin and decarboxylated sperm 4-carboxyglutamic acid-containing (Gla-)protein. The results obtained demonstrated that liver and vessel wall carboxylase may be regarded as isoenzymes with different substrate specificities. The newly discovered enzyme is the first vitamin K-dependent carboxylase which shows an absolute substrate specificity: FLEEL and decarboxylated osteocalcin were good substrates for vessel wall carboxylase, but decarboxylated fragment 13-29 and decarboxylated sperm Gla-protein were not carboxylated at all.     

Evidence for a radical mechanism of halogenation of monochlorodimedone catalyzed by chloroperoxidase

A radical species of monochlorodimedone has been characterized by its high reactivity with molecular O2. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous H2O2 only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of H2O2 added and O2 consumed. However, with excess Br- and horseradish peroxidase, or high Br- or Cl- and chloroperoxidase, a 1:1 stoichiometry between H2O2 and substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per H2O2. Moreover, in the absence of halide, at substrate:H2O2 ratios greater than 2.0, chloroperoxidase catalyzed significant O2 uptake; this enzyme-dependent autoxidation of monochlorodimedone also occurred in the presence of Cl- or Br-, when H2O2 was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Commun. 116, 873-879], strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOCl formation catalyzed by chloroperoxidase are considered.     

On the mechanism of chlorination by chloroperoxidase

Spectral-scan results obtained on the millisecond time scale are reported for reactions of chloroperoxidase with peracetic acid and chloride ion in both the presence and the absence of monochlorodimedone. A multimixing experiment is performed in which stoichiometric amounts of chloroperoxidase and peracetic acid are premixed for 0.7 s before the resultant compound I is reacted with chloride ion. The combined results show that the only detectable enzyme intermediate species is compound I (except in very late stages of the reaction), that the disappearance of compound I is accelerated by the presence of chloride ion, and that it is further accelerated if both chloride and monochlorodimedone are present. It is concluded that compound I is an obligate intermediate species in the reaction. Experiments are performed on the reaction of monochlorodimedone with hypochlorous acid in both the presence and the absence of added chloride ion, but in the absence of chloroperoxidase. The presence of chloride ion greatly accelerates the reaction rate apparently by setting off a chlorine chain reaction. This reaction would be important in the enzyme-catalyzed reaction if hypochlorous acid were liberated into the solution. A careful analysis of steady-state kinetic results shows that in the chlorination of monochlorodimedone at least, liberation of free hypochlorous acid is not important in the enzyme-catalyzed pathway. Rather the reaction proceeds from compound I to formation of iron(III)-OCl by chloride ion addition to the ferryl oxygen atom. This obligate intermediate species then chlorinates the substrate. It is well described as enzyme-activated hypochlorous acid, in which replacement of the proton in HOCl by the heme iron ion produces a Cl+ species of great potency. Thus the enzyme controls chlorination of monochlorodimedone rather than unleashing an uncontrolled chain reaction in which it would be rapidly destroyed.     

Chloroperoxidase-catalyzed enantioselective oxidation of methyl phenyl sulfide with dihydroxyfumaric acid/oxygen or ascorbic acid/oxygen as oxidants

The chloroperoxidase catalyzed oxidation of methyl phenyl sulfide to (R)-methyl phenyl sulfoxide was investigated, both in batch and membrane reactors, using as oxidant H2O2, or O2 in the presence of either dihydroxyfumaric acid or ascorbic acid. The effects of pH and nature and concentration of the oxidants on the selectivity, stability, and productivity of the enzyme were evaluated. The highest selectivity was displayed by ascorbic acid/O2, even though the activity of chloroperoxidase with this system was lower than that obtained with the others. When the reaction was carried out in a membrane reactor, it was possible to reuse the enzyme for several conversion cycles. The results obtained with ascorbic acid/O2 and dihydroxyfumaric acid/O2 as oxidants do not seem to be compatible with either a mechanism involving hydroxyl radicals as the active species or with the hypothesis that oxidation occurs through the initial formation of H2O2. Copyright 1999 John Wiley & Sons, Inc.     

The chlorination of barbituric acid and some of its derivatives by chloroperoxidase

Barbituric acid and some of its derivatives are presented as new substrates for the chloroperoxidase from Caldariomyces fumago. These compounds are rapidly converted to the 5-chloro or 5,5-dichloro derivatives, in very high yield. The reaction path is discussed and the kinetics of the reactions are investigated. It is shown that neither the concentration nor the structure of the organic substrate has any influence on the rate of halogenation. The enzymatic chlorination of 1-methyl-5-phenylbarbituric acid does not proceed in a stereoselective manner. The results are compared with the present theories concerning the enzymatic reaction mechanism, and the current research on this topic is evaluated. The available data do not as yet permit a definitive choice of reaction mechanism.     

Altering catalytic properties of 3-chlorocatechol-oxidizing extradiol dioxygenase from Sphingomonas xenophaga BN6 by random mutagenesis

The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.     

Catalytic properties of rice alpha-oxygenase. A comparison with mammalian prostaglandin H synthases

Long-chain fatty acids can be metabolized to C(n)(-1) aldehydes by alpha-oxidation in plants. The reaction mechanism of the enzyme has not been elucidated. In this study, a complete nucleotide sequence of fatty acid alpha-oxygenase gene in rice plants (Oryza sativa) was isolated. The deduced amino acid sequence showed some similarity with those of mammalian prostaglandin H synthases (PGHSs). The gene was expressed in Escherichia coli and purified to apparently homogeneous state. It showed the highest activity with linoleic acid and predominantly formed 2-hydroperoxide of the fatty acid (C(n)), which is then spontaneously decarboxylated to form corresponding C(n)(-1) aldehyde. With linoleic or linoleic acids as a substrate, rice alpha-oxygenase formed no product having a lambda(max) at approximately 234 nm, which indicated that the enzyme could not oxygenize the pentadiene system in the substrate. The spectroscopic feature of the purified enzyme in its ferrous state is similar to that of mammalian PGHS, whereas that of dithionite-reduced state showed significant difference. Site-directed mutagenesis revealed that His-158, Tyr-380, and Ser-558 were essential for the alpha-oxygenase activity. These residues are conserved in PGHS and known as a heme ligand, a source of a radical species to initiate oxygenation reaction and a residue involved in substrate binding, respectively. This finding suggested that the initial step of the oxygenation reaction in alpha-oxygenase has a high similarity with that of PGHS. The rice alpha-oxygenase activity was inhibited by imidazole but hardly inhibited by nonsteroidal anti-inflammatory drugs, such as aspirin, ibuprofen, and flurbiprofen, which are known as typical PGHS inhibitors. In addition, peroxidase activity could not be detected with alpha-oxygenase when palmitic acid 2-hydroperoxide was used as a substrate. From these findings, the catalytic resemblance between alpha-oxygenase and PGHS seems to be evident, although there still are differences in their substrate recognitions and peroxidation activities.     

Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems

Although salicylates have been used for centuries as treatment of inflammatory diseases, the mechanism of action of these drugs is still not clear. Aspirin (acetylsalicylic acid) and other nonsteroidal anti-inflammatory drugs (NSAID) inhibit prostaglandin biosynthesis, a property that appears to explain part of their anti-inflammatory activity. However, this mechanism does not appear to explain the anti-inflammatory properties of salicylic acid, which is a major metabolite of ASA in vivo. Results of prior studies in our laboratory have established that benzoic acid, the parent compound of the salicylate group of drugs, is decarboxylated and hydroxylated by the hydroxyl free radical (OH.) produced by stimulated granulocytes. These observations suggested that salicylates might be similarly metabolized by granulocytes. If so, the capacity of salicylates to rapidly react with OH. might relate directly to their known anti-inflammatory properties. Preliminary experiments established that salicylic acid and aspirin were decarboxylated by the hydroxyl free radical generated by the enzyme system xanthine-xanthine oxidase. We then studied the metabolism of salicylates by human granulocytes. Unstimulated granulocyte suspensions did not oxidize ASA or salicylic acid. However, suspensions stimulated by opsonized zymosan particles rapidly oxidized both substrates in pharmacological concentrations. The rate of oxidation of salicylic acid was 16-fold higher than benzoic acid, whereas the rate of oxidation of ASA was four-fold higher. The reaction was oxygen dependent and could be inhibited by known hydroxyl scavengers, particularly dimethylthiourea. The reaction could also be inhibited by superoxide dismutase and azide, indicating that O-2 and heme or an iron-dependent enzyme were required for the reaction. The reaction was not impaired by compounds known to react with the HOCL and the chloramines generated by stimulated PMN. Furthermore, salicylic acid in high concentrations did not impair the HMPS pathway, the production of O-2 or the production of H2O2 by granulocytes. These data provide evidence that salicylates are rapidly oxidized by the hydroxyl free radical produced by granulocytes and not O-2, H2O2, or HOCL. This capacity of salicylates to react rapidly and selectively react with OH. may directly relate to their anti-inflammatory properties. In addition, results of our experiments indicate that stimulated granulocytes acquire the capacity to metabolize these drugs. Therefore, several metabolites of salicylates may be produced at a site of inflammation, all of which may have altered biological activity compared with the parent compound.     

beta-Decarboxylation of L-aspartic acid: a metal chelate-catalyzed reaction.

beta-decarboxylation of L-aspartic acid was observed in the system, pyridoxal: L-aspartic acid:aluminum(III), 1:100:1 when heated at 80 degrees for three hours. This reaction was followed by electronic spectroscopy and showed quantitative conversion of pyridoxal to pyridoxamine indicating decarboxylation of the ketimine. alpha-Methyl-L-aspartic acid was not decarboxylated indicating the presence of the alpha-proton and prior transamination as requirements for decarboxylation. When pyridoxamine and oxalo-2-propionic acid were reacted at pD 4.60, product analysis by nmr showed the presence of pyridoxamine and alpha-ketobutyric acid, indicating hydrolysis of the decarboxylated ketimine. Decarboxylation was fast compared to spontaneous decarboxylation. A mechanism is proposed for non-enzymatic decarboxylation and the previously suggested mechanism for the inactivation of the enzyme aspartate beta-decarboxylase is discussed.     

Kinetics of the oxidation of ascorbic acid, ferrocyanide and p-phenolsulfonic acid by chloroperoxidase compounds I and II

For the first time elementary reactions involving chloroperoxidase compounds I and II have been investigated. A multi-mixing stopped-flow apparatus was used to study the kinetics of the reactions of compounds I and II with ascorbic acid, ferrocyanide and p-phenolsulfonic acid. The second-order rate constants of the reactions of both compounds with all three substrates were determined between pH 3 and pH 7. In all cases the rate constants decrease with increasing pH. The reactions of p-phenolsulfonic acid are influenced by a catalytically important group on both compounds I and II with a pKa of 3.7 +/- 0.2. With ascorbic acid and ferrocyanide as substrates, a decrease in rate was observed upon ionization of the substrate. Comparisons with horseradish peroxidase show that chloroperoxidase is a much less efficient peroxidatic enzyme. The kinetic data were used to calculate the percentage composition of the mixture of chloroperoxidase species which contribute to the spectra measured during the turnover with ascorbate as substrate.     

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.     

Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.     

Mechanisms of 1,3-butadiene oxidations to butadiene monoxide and crotonaldehyde by mouse liver microsomes and chloroperoxidase

NADPH-dependent oxidation of 1,3-butadiene by mouse liver microsomes or H2O2-dependent oxidation by chloroperoxidase produced both butadiene monoxide and crotonaldehyde; methyl vinyl ketone and 2,3- and 2,5- dihydrofuran were not detected. The crotonaldehyde to butadiene monoxide ratio remained constant over time in both the microsomal and the chloroperoxidase reactions; however, much more crotonaldehyde was produced by chloroperoxidase than microsomes; crotonaldehyde was not detected when reference samples of butadiene monoxide were used in control incubations containing NADPH and microsomes or H2O2 and chloroperoxidase. Moreover, incubations of 1,3-butadiene with horseradish peroxidase and H2O2, or microsomes and H2O2 or arachidonic acid did not result in the oxidation of 1,3-butadiene. In microsomes, metabolite formation was dependent on incubation time, NADPH, and protein concentrations and did not change when the 1,3-butadiene pressure was varied between 24 and 52 cm Hg. Inclusion of the cytochrome P450 inhibitor 1-benzylimidazole inhibited 1,3-butadiene metabolism, but inclusion of KCN, catalase, or superoxide dismutase had no effect. These results support the role of cytochrome P450 in 1,3-butadiene oxidation by mouse liver microsomes. The formation of crotonaldehyde but not methyl vinyl ketone by cytochrome P450 or chloroperoxidase indicates regioselectivity in the oxygen transfer from the hemoproteins to 1,3-butadiene. The intermediates formed may undergo either ring closure to form butadiene monoxide or a hydrogen shift to form 3-butenal which tautomerizes to produce crotonaldehyde. Evidence for this tautomerization was obtained by the finding that 3-buten-1-ol, an alternative precursor of 3-butenal, was oxidized to crotonaldehyde under incubation conditions similar to that used for 1,3-butadiene.     

Geranylgeranyl reductase involved in the biosynthesis of archaeal membrane lipids in the hyperthermophilic archaeon Archaeoglobus fulgidus

Complete saturation of the geranylgeranyl groups of biosynthetic intermediates of archaeal membrane lipids is an important reaction that confers chemical stability on the lipids of archaea, which generally inhabit extreme conditions. An enzyme encoded by the AF0464 gene of a hyperthermophilic archaeon, Archaeoglobus fulgidus, which is a distant homologue of plant geranylgeranyl reductases and an A. fulgidus menaquinone-specific prenyl reductase [Hemmi H, Yoshihiro T, Shibuya K, Nakayama T, & Nishino T (2005) J Bacteriol187, 1937-1944], was recombinantly expressed and purified, and its geranylgeranyl reductase activity was examined. The radio HPLC analysis indicated that the flavoenzyme, which binds FAD noncovalently, showed activity towards lipid-biosynthetic intermediates containing one or two geranylgeranyl groups under anaerobic conditions. It showed a preference for 2,3-di-O-geranylgeranylglyceryl phosphate over 3-O-geranylgeranylglyceryl phosphate and geranylgeranyl diphosphate in vitro, and did not reduce the prenyl group of respiratory quinones in Escherichia coli cells. The substrate specificity strongly suggests that the enzyme is involved in the biosynthesis of archaeal membrane lipids. GC-MS analysis of the reaction product from 2,3-di-O-geranylgeranylglyceryl phosphate proved that the substrate was converted to archaetidic acid (2,3-di-O-phytanylglyceryl phosphate). The archaeal enzyme required sodium dithionite as the electron donor for activity in vitro, similarly to the menaquinone-specific prenyl reductase from the same anaerobic archaeon. On the other hand, in the presence of NADPH (the preferred electron donor for plant homologues), the enzyme reaction did not proceed.     

Chlorinations catalyzed by chloroperoxidase occur via diffusible intermediate(s) and the reaction components play multiple roles in the overall process

The chlorination mechanism of the fungal enzyme chloroperoxidase (CPO) has been debated for (1) active site chlorination and (2) diffusible species mediated chlorination. Based upon the conversion of approximately 35 different substrates belonging to different reactive groups, it was found that substrate dimensions and topography had no pronounced effect on rates of CPO chlorination reaction. Epoxidation of indene was dependent on its concentration where as chlorination was not. Also, effective conversion was seen in the chlorination mixture for substrates that could not be epoxidized or sulfoxidized. Some insoluble substrates and certain molecules that exceeded the active site dimensions were chlorinated at rates comparable to the rates required for CPO's more natural substrate, monochlorodimedone. By terminating the enzymatic reaction with an active site ligand (azide), the amount of diffusible species was correlated to CPO in the reaction mixture. The preferential utilization of a substrate, earlier attributed to the active site, is found to be due to the specificity afforded by the reaction environment. It was found that the reaction medium components of peroxide, chloride and hydronium ions affected the reaction rates through varying roles in the enzymatic and non-enzymatic process. Besides these experimental evidences, key mechanistic and kinetic arguments are presented to infer that the final chlorine transfer occurs outside the active site via a diffusible species.     

Peroxidase-catalyzed N-demethylation reactions. Substrate deuterium isotope effects

Deuterium isotope effects on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase were determined using N,N-di-(trideuteromethyl)aniline. The isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction supported by ethyl hydroperoxide was 1.42 +/- 0.31. The isotope effects on the Vmax for the horseradish peroxidase-catalyzed reaction supported by ethyl hydroperoxide and hydrogen peroxide were 1.99 +/- 0.39 and 4.09 +/- 0.27, respectively. Isotope effects ranging from 1.76 to 5.10 were observed on the Vmax/Km for the hydroperoxide substrate (i.e. the second order rate constant for the reaction of the hydroperoxide with the peroxidase to form compound I) in both enzyme systems when the N-methyl groups of N,N-dimethylaniline were deuterated. These results are not predicted by the simple ping-pong kinetic model for peroxidase-catalyzed N-demethylation reactions. The data are most simply explained by a mechanism involving the transfer of deuterium (or hydrogen) from N,N-dimethylaniline to the enzyme during catalysis. The deuterium must subsequently be displaced from the enzyme by the hydroperoxide, causing the observed isotope effects.     

Oxidation-reduction potential measurements on chloroperoxidase and its complexes.

The oxidation-reduction potential of chloroperoxidase, an enzyme which catalyzes peroxidative chlorination, bromination, and iodination reactions, has been investigated. In addition to catalyzing biological halogenation reactions, chloroperoxidase is unusual in that the carbon monoxide complex of ferrous chloroperoxidase shows the typical long wavelength Soret absorption associated with P-450 hemoproteins. The pH dependence of the chloroperoxidase oxidation-reduction potential shows a discontinuity around pH 4.7. Similarly, measurements of the affinity of ferrous chloroperoxidase for carbon monoxide monitored both by spectroscopic and potentiometric titration exhibit a discontinuity in the pH 4.7 region. Oxidation-reduction potential measurements on chloroperoxidase in a CO atmosphere also show a discontinuous pH profile. These results suggest that ferrous chloroperoxidase undergoes reversible modification at low pH and that these changes are reflected in the oxidation-reduction potential. The oxidation-reduction potential of chloroperoxidase at pH 6.9 is - 140 mV, close to that measured for cytochrome P-450cam in the presence of substrate. The oxidation-reduction potential of chloroperoxidase at pH 2.7, the pH optimum for enzymatic chlorination, is +150 mV. The oxidation-reduction potentials of the halide complexes of chloroperoxidase (chloride, bromide, and iodide) are essentially identical with the potential measurements on the native enzyme. These observations suggest that, although halide anions bind to the enzyme, they probably do not bind as an axial ligand to the heme ferric iron.     

Purification and kinetic properties of ox brain histamine N-methyltransferase.

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.     

Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme)

The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle.