Mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine

Functionalization of the beta-carbon of phenethylamines has been shown to produce a new class of substrate/inhibitor of dopamine beta-monooxygenase. Whereas both beta-hydroxy- and beta- chlorophenethylamine are converted to alpha-aminoacetophenone at comparable rates, only the latter conversion is accompanied by concomitant enzyme inactivation ( Klinman , J. P., and Krueger , M. (1982) Biochemistry 21, 67-75). In the present study, the nature of the reactive intermediates leading to dopamine beta-monooxygenase inactivation by beta- chlorophenethylamine has been investigated employing kinetic deuterium isotope effects and oxygen- 18 labeling as tools. Mechanistically significant findings presented herein include: 1) an analysis of primary deuterium isotope effects on turnover, indicating major differences in rate-determining steps for beta-chloro- and beta- hydroxyphenethylamine hydroxylation, Dkcat = 6.1 and 1.0, respectively; 2) evidence that dehydration of the gem-diol derived from oxygen- 18-labeled beta- hydroxyphenethylamine hydroxylation occurs in a random manner, attributed to dissociation of enzyme-bound gem-diol prior to alpha-aminoacetophenone formation; 3) the observation of a deuterium isotope effect for beta- chlorophenethylamine inactivation, Dkinact = 3.7, implicating C--H bond cleavage in the inactivation process; and 4) the demonstration that alpha-aminoacetophenone can replace ascorbic acid as exogenous reductant in the hydroxylation of tyramine. As discussed, these findings support the intermediacy of enzyme-bound alpha-aminoacetophenone in beta- chlorophenethylamine inactivation, and lead us to propose an intramolecular redox reaction to generate a ketone-derived radical cation as the dopamine beta-monooxygenase-inactivating species.     

Magnitude of intrinsic isotope effects in the dopamine beta-monooxygenase reaction

Intrinsic primary hydrogen isotope effects (kH/kD) have been obtained for the carbon-hydrogen bond cleavage step catalyzed by dopamine beta-monooxygenase. Irreversibility of this step is inferred from the failure to observe back-exchange of tritium from TOH into substrate under conditions of dopamine turnover; this result cannot be due to solvent inaccessibility at the enzyme active site, since we will demonstrate [Ahn, N., & Klinman, J. P. (1983) Biochemistry (following paper in this issue)] that a solvent-derived proton or triton must be at the enzyme active site prior to substrate activation. As shown by Northrop [Northrop, D. B. (1975) Biochemistry 14, 2644], for enzymatic reactions in which the carbon-hydrogen bond cleavage step is irreversible, comparison of D(V/K) to T(V/K) allows an explicit solution for kH/kD. Employing a double-label tracer method, we have been able to measure deuterium isotope effects on Vmax/Km with high precision, D(V/K) = 2.756 +/- 0.054 at pH 6.0. The magnitude of the tritium isotope effect under comparable experimental conditions is T(V/K) = 6.079 +/- 0.220, yielding kH/kD = 9.4 +/- 1.3. This result was obtained in the presence of saturating concentrations of the anion activator fumarate. Elimination of fumarate from the reaction mixture leads to high observed values for isotope effects on Vmax/Km, together with an essentially invariant value for kH/kD = 10.9 +/- 1.9. Thus, the large disparity between isotope effects, plus or minus fumarate, cannot be accounted for by a change in kH/kD, and we conclude a role for fumarate in the modulation of the partitioning of enzyme-substrate complex between catalysis and substrate dissociation. On the basis of literature correlations of primary hydrogen isotope effects and the thermodynamic properties of hydrogen transfer reactions, the very large magnitude of kH/kD = 9.4-10.9 for dopamine beta-monooxygenase suggests an equilibrium constant not very far from unity for the carbon-hydrogen bond cleavage step. This feature, together with the failure to observe re-formation of dopamine from enzyme-bound intermediate or product and overall rate limitation of enzyme turnover by product release, leads us to propose a stepwise mechanism for norepinephrine formation from dopamine in which carbon-hydrogen bond cleavage is uncoupled from the oxygen insertion step.     

Olefin oxygenation and N-dealkylation by dopamine beta-monooxygenase: catalysis and mechanism-based inhibition

In an initial communication [May, S. W., Mueller, P. W., Padgette, S. R., Herman, H. H., & Phillips, R. S. (1983) Biochem. Biophys. Res. Commun. 110, 161-168], we reported that 1-phenyl-1-(aminomethyl)ethene hydrochloride (PAME) is an olefinic substrate for dopamine beta-monooxygenase (DBM; EC 1.14.17.1) which inactivates the enzyme in an apparent mechanism-based manner. The present study further characterizes this reaction. The inactivation reaction yields kinact = 0.23 min-1 at pH 5.0 and 37 degrees C and is strictly dependent on reductant (ascorbate) and oxygen. The DBM/PAME substrate reaction (apparent kcat = 14 s-1), shown to be stimulated by fumarate, gives the corresponding epoxide as product, identified by derivatization with 4-(p-nitrobenzyl)pyridine. However, the lack of DBM inhibition by alpha-methylstyrene oxide, and the observation of identical PAME/DBM inactivation rates in the absence and presence of preformed enzymatic PAME epoxide, indicates that free epoxide is not the inactivating species. A structure-activity study revealed that 4-hydroxylation of PAME (to give 4-HOPAME) increases both kinact (0.81 min-1) and apparent kcat (56 s-1) values, while 3-hydroxylation (to give 3-HOPAME) greatly diminishes inactivation activity while retaining substrate activity (apparent kcat = 47 s-1). 4-Hydroxy-alpha-methylstyrene was found to be a DBM inhibitor (kinact = 0.53 min-1) with weak substrate activity (apparent kcat = 0.71 s-1), while 3-hydroxy-alpha-methylstyrene and alpha-(cyanomethyl) styrene were found not to exhibit detectable DBM substrate activity and only weak inhibitory activity. 3-Phenylpropargylamine hydrochloride showed no detectable DBM substrate activity but rapidly inactivated the enzyme. A new substrate activity for DBM was discovered, N-dealkylation of N-phenylethylenediamine and N-methyl-N-phenylethylenediamine, and the lack of O-dealkylation activity with phenyl 2-aminoethyl ether and 4-hydroxyphenyl 2-aminoethyl ether indicates that DBM N-dealkylation proceeds via initial one-electron abstraction from the benzylic nitrogen heteroatom. With this new substrate and inhibitor reactivity information in hand, along with the other known substrate reactions, a DBM oxygenation mechanism analogous to that for cytochrome P-450 is proposed.     

A monoclonal antibody to dopamine beta-monooxygenase: detection of biosynthetic intermediates

A monoclonal antibody to dopamine beta-monooxygenase (DBH) has been produced by an in vitro immunization technique. This antibody has been found to react with an epitope common to both soluble DBH (SDBH) and membrane-bound DBH (MDBH). A single CNBr fragment contains this antigenic site. Examination of electrophoretograms of chromaffin granule membrane and lysate by the immunoblot procedure revealed additional complexity. A 58-KDa polypeptide in low abundance was stained which could not be detected with the polyclonal antiserum. Chemical deglycosylation of SDBH produces two new polypeptides of 67 and 58 KDa. The entirety of this data suggests that the 58-KDa band is an unglycosylated form of DBH, proteolytically cleaved after biosynthesis.     

Secondary isotope effects and structure-reactivity correlations in the dopamine beta-monooxygenase reaction: evidence for a chemical mechanism

The chemical mechanism of hydroxylation, catalyzed by dopamine beta-monooxygenase, has been explored with a combination of secondary kinetic isotope effects and structure-reactivity correlations. Measurement of primary and secondary isotope effects on Vmax/Km under conditions where the intrinsic primary hydrogen isotope effect is known allows calculation of the corresponding intrinsic secondary isotope effect. By this method we have obtained an alpha-deuterium isotope effect, Dk alpha = 1.19 +/- 0.06, with dopamine as substrate. The beta-deuterium isotope effect is indistinguishable from one. The large magnitude of Dk alpha, together with our previous determination of a near maximal primary deuterium isotope effect of 9.4-11, clearly indicates the occurrence of a stepwise process for C-H bond cleavage and C-O bond formation and hence the presence of a substrate-derived intermediate. To probe the nature of this intermediate, a structure-reactivity study was performed by using a series of para-substituted phenylethylamines. Deuterium isotope effects on Vmax and Vmax/Km parameters were determined for all of the substrates, allowing calculation of the rate constants for C-H bond cleavage and product dissociation and dissociation constants for amine and O2 loss from the enzyme-substrate ternary complex. Multiple regression analysis yielded an electronic effect of p = -1.5 for the C-H bond cleavage step, eliminating the possibility of a carbanion intermediate. A negative p value is consistent with formation of either a radical or a carbocation; however, a significantly better correlation is obtained with sigma p rather than sigma p+, implying formation of a radical intermediate via a polarized transition state. Additional effects determined from the regression analyses include steric effects on rate constants for substrate hydroxylation and product release and on KDamine, consistent with a sterically restricted binding site, and a positive electronic effect of p = 1.4 on product dissociation, ascribed to a loss of product from an enzyme-bound Cu(II)-alkoxide complex. These results lead us to propose a mechanism in which O-O homolysis [from a putative Cu(II)-OOH species] and C-H homolysis (from substrate) occur in a concerted fashion, circumventing the formation of a discrete, high energy oxygen species such as hydroxyl radical. The substrate and peroxide-derived radical intermediates thus formed undergo a recombination, kinetically limited by displacement of an intervening water molecule, to give the postulated Cu(II)-alkoxide product complex.     

Purification of membrane-bound dopamine beta-monooxygenase from chromaffin granules: relation to soluble dopamine beta-monooxygenase

Previous studies have indicated that α-d-1-fluoroglucose is a glycosyl donor for glucosyl transferases (5, 6) including dextransucrases formed by Leuconostoc and Streptococcus mutans. The present report confirms these observations with dextransucrase isolated from S. sanguis and conclusively establishes the details of this reaction as well as proving that mechanism of fluoroglucose transfer is comparable to that glucosyl transfer from sucrose. A new procedure for monitoring the reaction is reported, and is based on the measurement of proton formation using the pH indicator, bromcresol purple. Production of F^? was found to be stoichiometric with proton production. Rate studies with the substrate indicate that α-1-fluoroglucose undergoes spontaneous hydrolysis, which is greatly increased in the presence of nucleophilic buffers. When [¹⁴C]maltose and α-1-fluoroglucose or [¹⁴C]α-1-fluoroglucose and maltose were incubated with dextransucrase, a series of oligosaccharide products was observed. The results indicate that the glucosyl moiety of α-1-fluoroglucose transferred to the acceptor. The nature of formation of the products are consistent with a series of precursor-product reactions. Product analysis of the saccharides by borohydride reduction analysis demonstrated that the glucosyl unit was added to the nonreducing end of maltose. When either [¹⁴C]fructose or [¹⁴C]-α-1-fluoroglucose were incubated with enzyme, a reaction was observed which was analogous to the isotopic-exchange reaction catalyzed by the enzyme in the presence of [¹⁴C]fructose and sucrose.     

Beta-lactamase inactivation by mechanism-based reagents

The mechanistic pathway followed by the E. coli RTEM beta-lactamase has been studied with a view to clarifying the mode of action of a number of recently discovered inactivators of the enzyme. There is clear evidence that the beta-lactamase-catalysed hydrolysis of the 7-alpha-methoxycephem, cefoxitin, proceeds via an acyl-enzyme intermediate. An analysis of the inactivation reactions of all the known beta-lactam derivatives that result in irreversible loss of enzyme activity permits the identification of three structural features required for a beta-lactamase inactivator. The application of these principles suggests a new group of mechanism-based inactivators of the enzyme: the sulphones of N-acyl derivatives of 6-beta-aminopenicillanic acid that are themselves poor substrates for the enzyme. These sulphones are powerful inactivators of the beta-lactamase.     

Mechanism of inhibition of dopamine beta-monooxygenase by quinol and phenol derivatives, as determined by solvent and substrate deuterium isotope effects.

The mechanism of interaction of quinols and phenols with dopamine beta-monooxygenase (D beta M) has been investigated. The ratio of quinone formation (from catechol) to oxygen consumption rises from a value of 1 in the presence of phenethylamine substrate to 2 in the absence of substrate. These results implicate quinol oxidation at both the reductant- and substrate-binding sites of D beta M. In the presence of saturating ascorbate, catechol and p-hydroquinol behave as mechanism-based inhibitors of D beta M, with partitioning ratios of turnover to inactivation of 21:1 and 41:1, respectively. Phenol is found to inactivate the enzyme in a manner similar to p-cresol, suggesting that the methyl group of p-cresol is not an essential component of enzyme inhibition. Solvent isotope effects on inactivation and turnover have been measured for various inactivators. Although the majority of these inhibitors, including catechol, p-hydroquinol, aniline, phenethylenediamine, and benzylhydrazine, are characterized by relatively small solvent isotope effects (1.5-2.5) on the inactivation rate constant (ki), solvent isotope effects on ki for phenol and p-cresol are 5.7 and 7.4, respectively. By contrast, solvent isotope effects on the turnover of p-cresol are almost unity. Using p-cresol-d7 as substrate, we observe D(kcat) = 5.2 and D(kcat/Km) = 3.1, while isotope effects on inactivation are D(ki) = 0.95 and D(ki/Ki) = 0.59. These results lead us to propose that inhibitors fall into two mechanistic classes, involving either one-electron oxidation to form radical cation intermediates (quinols) or hydrogen atom abstraction (phenols).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism-based inhibition of dopamine beta-monooxygenase by aldehydes and amides

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which enzyme-bound alpha-aminoacetophenone is generated, followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the enzyme inhibitory species (Mangold, J. B., and Klinman, J. P. (1984) J. Biol. Chem. 259, 7772-7779). If correct, additional compounds capable of producing enzyme-bound (formula; see text) reductant should inhibit dopamine beta-monooxygenase. Phenylacetaldehyde was chosen to test this model, since beta-hydroxyphenylacetaldehyde is expected to function as a reductant in a manner analogous to alpha-aminoacetophenone. Phenylacetaldehyde exhibits the properties of a mechanism-based inhibitor. Kinetic parameters are comparable to beta-chlorophenethylamine under both initial velocity and inactivation conditions. Since phenylacetaldehyde bears little resemblance to beta-chlorophenethylamine, its analogous inhibitory action provides support for an intramolecular redox reaction (via beta-hydroxyphenylacetaldehyde oxidation to a radical cation) in dopamine beta-monooxygenase inactivation. beta-Hydroxyphenylacetaldehyde was identified as the enzymatic product of phenylacetaldehyde turnover. As predicted, this product behaves both as a time-dependent inhibitor of dopamine beta-monooxygenase and as an electron donor in enzyme-catalyzed hydroxylation of tyramine to octopamine. Phenylacetamide and p-hydroxyphenylacetamide are also found to be mechanism-based inhibitors of dopamine beta-monooxygenase. In this case the product of hydroxylation (beta-hydroxyphenylacetamide) is redox inactive and, therefore, is unable to function as either a reductant or an inhibitor. Thus, mechanism-based inhibitors are divided into two types: type I, which undergoes hydroxylation prior to inactivation, and type II, which only requires hydrogen atom abstraction. A general mechanism for dopamine beta-monooxygenase inactivation is described, in which a common mechanistic radical intermediate is formed from both pathways.     

Mechanism of modulation of dopamine beta-monooxygenase by pH and fumarate as deduced from initial rate and primary deuterium isotope effect studies

Dopamine beta-monooxygenase catalyzes a reaction in which 2 mol of protons are consumed for each turnover of substrate. Studies of the pH dependence of initial rate parameters (Vmax and Vmax/Km) and their primary hydrogen isotope effects show that at least two ionizable residues are involved in catalysis. One residue (B1, pK = 5.6-5.8) must be protonated prior to the carbon-hydrogen bond cleavage step, implying a role for general-acid catalysis in substrate activation. A second protonated residue (B2, pK = 5.2-5.4) facilitates, but is not required for, product release. Recent measurement of the intrinsic isotope effect for dopamine beta-monoxygenase [Miller, S. M., & Klinman, J. P. (1983) Biochemistry (preceding paper in this issue)] allows an analysis of the pH dependence of rate constant ratios and in selected instances individual rate constants. We demonstrate large changes in the rate-determining step as well as an unprecedented inversion in the kinetic order of substrate release from ternary complex over an interval of 2 pH units. Previously, fumarate has been used in dopamine beta-monooxygenase assays because of its property of enzyme activation. Studies of the pH behavior in the presence of saturating concentrations of fumarate have shown two causes of the activation: (i) fumarate perturbs the pK of B1 to pK = 6.6-6.8 such that the residue remains protonated and the enzyme optimally active over a wider pH range; (ii) fumarate decreases the rate of dopamine release from the ternary enzyme-substrate complex, increasing the equilibrium association constant for dopamine binding. Both effects are consistent with a simple electrostatic stabilization of bound cationic charges by the dianionic form of fumarate.     

Use of isotope effects to elucidate enzyme mechanisms

The chemical bond breaking steps are normally not rate limiting for enzymatic reactions. However, comparison of deuterium and tritium isotope effects on the same reaction, especially when coupled with 13C isotope effects for the same step measured with deuterated as well as unlabeled substrates, allows calculation of the intrinsic isotope effects on the bond breaking steps and thus a determination of the commitments to catalysis for the reactants. The variation in observed isotope effects as a function of reactant concentration can be used to determine kinetic mechanisms, while the pH variation of isotope effects can determine the stickiness of the reactants and which portions of the reactant mechanism are pH dependent. Finally the size of primary and secondary intrinsic isotope effects can be used to determine transition state structure.     

Purification and characterization of rat dopamine beta-monooxygenase and monoclonal antibodies to the enzyme

Dopamine beta-monooxygenase was extensively purified from rat adrenal. The specific activity of the final preparation was approx. 1500 nmol/min per mg protein, which was much higher than the highest yet reported. As judged by gel filtration on Ultrogel AcA22, SDS-polyacrylamide gel electrophoresis, and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 88 000. The isoelectric point of the enzyme was estimated to be pH 6.6 in the presence of 8 M urea. Spleen cells from BALB/c mice immunized with rat dopamine beta-monooxygenase were fused to P3-X63-Ag8-653 mouse myeloma cells. From 55 hybrid cells, 10 stable clones secreting anti-dopamine beta-monooxygenase antibody were obtained. Antibody from one clone was coupled to CNBr-activated Sepharose 4B and the monoclonal antibody-Sepharose was shown to be very useful to isolate rat dopamine beta-monooxygenase from crude preparations.     

An acetylenic mechanism-based inhibitor of dopamine beta-hydroxylase

The catalytic action of dopamine beta-hydroxylase on 1-phenyl-1-propyne results in concomitant loss of enzyme activity. At pH 5.5 and 25 degrees C, 1-phenyl-1-propyne inactivates dopamine beta-hydroxylase in a mechanism-based fashion. The inactivation rate is first-order, follows saturation kinetics, and is strictly dependent on catalysis (oxygen and ascorbate are essential). The inactivation rate of saturating 1-phenyl-1-propyne (kinact) increases from 0.08 to 0.22 min-1 when the oxygen saturation increases from 21 to 100%, respectively. Inactivation also requires a copper-containing catalytically competent enzyme. Tyramine and norepinephrine (respectively, substrate and product of the normal catalytic reaction) protect against inactivation, and no regain of enzyme activity occurs after prolonged dialysis. Experiments with ether-extracted incubation solutions (+/- enzyme) showed no difference in their gas chromatography-mass spectral patterns implying that inactivation of dopamine beta-hydroxylase by 1-phenyl-1-propyne occurs through a kinetic process with a partition ratio (kcat/kinact) equal to or near 1. Thus, this acetylenic substrate analog appears to be a very efficient mechanism-based inhibitor of dopamine beta-hydroxylase. We propose that inactivation of this enzyme by 1-phenyl-1-propyne proceeds by formation of a reactive intermediate that occurs prior to product formation and that alkylates an amino acid residue at the active site of the enzyme.     

Relation between the membrane and soluble forms of dopamine beta-monooxygenase

The quantitative ratio of membrane-bound and soluble forms of dopamine beta-monooxygenase from chromaffin granules obtained under different experimental conditions was determined. The amount of the membrane-bound form of dopamine beta-monooxygenase made up to no less than 60% of the total enzyme pool, when the granules were obtained and lyzed in the presence of pepstatin, phenylmethylsulfonyl fluoride, N-ethylmaleimide and catalase. In the absence of protectors practically all the enzyme can be obtained in the soluble form without detergent treatment. The effects of some ionic and nonionic detergents on the enzymatic activity of both forms of dopamine beta-monooxygenase were studied. No inhibition of dopamine beta-monooxygenase by 2% octyl glucoside or 1% Triton X-100 was observed. A comparative analysis of specific activities, subunit compositions, antigenic and physico-chemical properties of membrane-bound and soluble forms of dopamine beta-monooxygenase was carried out.     

Activation of dopamine beta-monooxygenase by external and internal electron donors in resealed chromaffin granule ghosts

Membrane ghosts derived from chromaffin vesicles of bovine adrenal medullas have been used to examine the mechanism of reduction of dopamine beta-monooxygenase in its compartmentalized state. The rate of the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine is greatly stimulated by the presence of ATP, reflecting substrate hydroxylation on the ghost interior subsequent to the active transport of dopamine. We demonstrate a 2-3-fold increase in the turnover rate for ghosts resealed with 0.2-2 mM potassium ferrocyanide, conditions leading to a slight decrease in the rate of dopamine transport. These data provide the first evidence that an intravesicular pool of reductant can activate dopamine beta-monooxygenase, as required by models in which vesicular ascorbate behaves as enzyme reductant. Although there is sufficient catecholamine (endogenous plus substrate) to keep internal ferrocyanide reduced in these experiments, an additional 2-3-fold increase in turnover occurs in the presence of 0.2-2 mM ascorbate on the ghost exterior. The magnitude of this activation is found to be constant at all concentrations of internal ferrocyanide (both below and above saturation), implying that reductants on opposite sides of the membrane behave independently. Replacement of ascorbate by potassium ferrocyanide as external reductant leads to almost identical results, and we are able to rule out an inward transport of dehydroascorbate as the source of activation by external ascorbate. We conclude that external reductants are capable of reducing membrane-bound dopamine beta-monooxygenase from the exterior face of the vesicle, either by direct reduction or through a membrane-bound mediator. It appears that two viable modes for reduction of dopamine beta-monooxygenase may exist in vivo, involving the reduction of membrane-bound enzyme by cytosolic ascorbate as well as the reduction of soluble enzyme by the pool of intravesicular ascorbate present in chromaffin vesicles.     

Mechanism of the insect enzyme, tyramine beta-monooxygenase, reveals differences from the mammalian enzyme, dopamine beta-monooxygenase

Tyramine beta-monooxygenase (TbetaM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TbetaM for comparison with the homologous mammalian enzymes, dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. A new and distinctive feature of TbetaM is very strong substrate inhibition that is dependent on the level of the co-substrate, O(2), and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of TbetaM, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of TbetaM is also reduced at high tyramine, leading us to propose the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells.     

Changes in dopamine uptake and developmental effects of dopamine receptor inactivation in the sea urchin

[³H]-dopamine ([³H]-DA) uptake was measured in the presence or absence of the catecholamine uptake inhibitor nomifensine in both unfertilized and fertilized eggs. Specific [³H]-DA uptake depended on time and [³H]-DA concentration; it was high in unfertilized eggs, declined 20–30 min after fertilization, and rose again during cleavage. Irreversible inactivation of dopamine receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in a complete loss of sensitivity of egg adenylate cyclase to dopamine stimulation. In fertilized eggs treated with EEDQ for 1 hr, restoration of adenylate cyclase activity sensitive to dopamine stimulation could be observed 4 hr after the end of treatment, thus suggesting the appearance of new dopamine receptors in cleaving eggs. Short-term EEDQ treatment on unfertilized eggs, although not impairing fertilization, resulted in cleavage inhibition; the same treatment carried out soon after fertilization, on the other hand, elicited no effect on development. On the contrary, in embryos subjected to continuous treatment with EEDQ, development was impaired independent of the stage at which the treatment was started. © 1995 Wiley-Liss, Inc.     

The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.