Mechanism-based inactivation of dopamine beta-hydroxylase by p-cresol and related alkylphenols

The mechanism-based inhibition of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding [DeWolf, W. E., Jr., & Kruse, L. I. (1985) Biochemistry 24, 3379]. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3096] p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate "activator" addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible, is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.     

3-Phenylpropenes as mechanism-based inhibitors of dopamine beta-hydroxylase: evidence for a radical mechanism

A series of ring-substituted 3-phenylpropenes has been examined as mechanism-based inhibitors for the copper protein dopamine beta-hydroxylase. p-HO-, p-CH3O-, m-HO-, m-CH3O-, p-Br-, and p-CN-substituted phenylpropenes all inactivate the enzyme under turnover conditions, requiring ascorbate and oxygen. Replacement of the benzylic hydrogens in 3-(p-hydroxyphenyl)propene with deuterium results in a kinetic isotope effect of 2.0 on kinact/KO2 but in no effect on the partition ratio, Vmax/kinact, consistent with a stepwise mechanism for hydrogen abstraction and oxygen insertion. The partition ratio is unchanged in the pH range from 4.5 to 7.1. Determination of the kinetics of inactivation and the partition ratios for each of these ring-substituted phenylpropenes has allowed determination of the respective V/KO2 values. A linear free energy plot of these values as a function of sigma+ gives a rho value of -1.2, while the partition ratios show only a slight decrease upon going electron-withdrawing groups. The results are consistent with a mechanism for dopamine beta-hydroxylase in which a hydrogen atom is abstracted to form a benzylic radical, which then partitions between hydroxylation and enzyme inactivation.     

Synthesis of several 2-substituted 3-(p-hydroxyphenyl)-1-propenes and their characterization as mechanism-based inhibitors of dopamine beta-hydroxylase

Three substrate analogs of dopamine beta-hydroxylase, viz. 2-X-3-(p-hydroxyphenyl)-1- propenes (where X = Br, Cl, H), have been synthesized, and all behave as substrates requiring O2 and ascorbate for the enzyme-catalyzed hydroxylation reaction. The products have been characterized by mass spectrometry as the respective 2-X-3-hydroxy-3-(p-hydroxyphenyl)-1- propenes . The relative kcat values for these compounds at pH 5.5, 0.25 mM O2 are 49 min-1 (2-H), 8.6 min-1 (2-Cl), and 7.0 min-1 (2-Br). All three compounds have the characteristics of mechanism-based inhibitors of dopamine beta-hydroxylase since incubation of enzyme with these compounds under turnover conditions leads to a time-dependent loss of activity. The kinact values at pH 5.5, 0.25 mM O2 are 0.08, 0.20, and 0.51 min-1, respectively, for the 2-Br-, 2-Cl-, and 2-H-substituted analogs. No reactivation was observed after exhaustive dialysis of enzyme inactivated by 2-Br-3-(p-hydroxyphenyl)-1-propene, suggesting irreversible inactivation of dopamine beta-hydroxylase.     

Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Alternate substrates of dopamine beta-hydroxylase. I. Kinetic investigations of benzyl cyanides as substrates and inhibitors

A series of benzyl cyanide analogs have been studied as substrates and inhibitors of dopamine beta-hydroxylase to extend our initial report (Baldoni, J. M., and Villafranca, J. J. (1980) J. Biol. Chem. 255, 8987-8990) which showed that p-hydroxybenzyl cyanide was a suicide substrate of dopamine beta-hydroxylase. Thus, the appVmax values for benzyl cyanide analogs decrease in the order p-OH greater than m-OH greater than H much greater than p-OCH3,m-OCH3; the m-OH, m-OCH3 and p-OCH3 analogs are competitive inhibitors versus tyramine in initial velocity studies. The Vmax values for tyramine and p-hydroxybenzyl cyanide are nearly identical at saturating O2 and ascorbate (pH 5.0, 37 degrees C) but the Km for O2 is 0.14 and 2.8 mM, respectively, with tyramine and p-hydroxybenzyl cyanide. Studies of the pH dependence of log V/K for tyramine show two pKa values of 5.2 and 5.8 while for m-hydroxybenzyl cyanide the values are 5.3 and 5.9. The log Vmax profile shows one pKa of 5.9 with tyramine as substrate. Thus, nearly identical enzymic groups are involved in binding and/or catalysis with these two substrates. All the benzyl cyanide analogs are suicide inactivators of dopamine beta-hydroxylase. With m-hydroxybenzyl cyanide, the partition between catalysis and inactivation (kcat/kinact) changed from approximately 600 to approximately 17 as the pH varied from 5.0 to 6.7. The log kinact versus pH profile shows one pKa value of 6.0, suggesting that an enzymic group must be deprotonated for maximal inactivation. Copper was essential for the suicide inactivation of dopamine beta-hydroxylase by benzyl cyanides and kinetic studies of partially inhibited dopamine beta-hydroxylase (approximately 50%) showed that inactive enzyme molecules were completely inactive. The following papers in this series discuss the partial reactivation of suicide-inhibited dopamine beta-hydroxylase and the stoichiometry of inactivation by benzyl cyanide analogs.     

The mechanism of inactivation of dopamine beta-hydroxylase by hydrazines

Dopamine beta-hydroxylase is inactivated by phenyl-, phenethyl-, benzyl-, and methylhydrazine, but not by hydrazine itself. With phenyl-, methyl-, and phenethylhydrazine, the rate of inactivation decreases in the presence of ascorbate and increases in the presence of tyramine. Reduction of the enzyme-bound copper occurs with all of the hydrazines tested. In the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone the carbon-centered radicals generated from each compound are trapped. This is consistent with reduction of the enzyme-bound copper by the hydrazine-containing compounds, resulting in formation of the hydrazine cation radical. Homolytic cleavage of the carbon-nitrogen bond then generates a carbon-centered radical which reacts with the enzyme, resulting in inactivation. Inactivation with [14C]phenylhydrazine results in the incorporation of 0.94 molecule of label per enzyme subunit. Benzylhydrazine behaves as a mechanism-based inhibitor of the enzyme. Both benzyl- and phenethylhydrazine are substrates for dopamine beta-hydroxylase. The second-order rate constant for inactivation of dopamine beta-hydroxylase by benzylhydrazine in the presence of ascorbate is increased about 4-fold when the benzylic hydrogens are replaced with deuterium. The apparent Vmax shows an observed deuterium kinetic isotope effect of 13 +/- 2. The partition ratio for product formation versus inactivation is 11-fold less for alpha,alpha-d2-benzylhydrazine. These results are interpreted in terms of a model where inactivation is due to abstraction of an electron from nitrogen instead of abstraction of a hydrogen atom from the benzylic carbon.     

Flavin-dependent alcohol oxidase from yeast. Studies on the catalytic mechanism and inactivation during turnover

The kinetic course of the reaction of methanol and deutero-methanol with FAD-dependent alcohol oxidase was investigated under single-turnover conditions [kred approximately equal to 15000 min-1 (1H3COH) and approximately equal to 4300 min-1 (2H3COH)] and multiple-turnover conditions [TNmax approximately equal to 6000 min-1 (1H3COH) and approximately equal to 3100 min-1 (2H3COH)]. A kinetic scheme for the overall catalytic mechanism is proposed, which is characterized by (1) formation of a Michaelis complex between enzyme and substrate, (2) the reductive step involving partly rate-limiting scission of the substrate C-H bond, (3) reaction of the complex of reduced enzyme and aldehyde with dioxygen, and (4) a significant contribution of the dissociation rate of product from its complex with reoxidized enzyme to the overall rate. Prolonged turnover of various alcohols, including methanol, results in progressive inactivation of the enzyme by two processes. In the absence of catalase the inactivation rate increases with time due to accumulation of hydrogen peroxide, which is a potent inactivator (Kd approximately equal to 1.6 mM; kinact approximately equal to 0.55 min-1). In the presence of catalase inactivation during turnover is much slower, the process showing pseudo-first-order kinetics (Kinact approximately equal to 0.6 mM; kinact approximately equal to 0.005 min-1 with methanol). The ratio kcat/kinact varies with different alcohols but is always greater than 10(5). Propargyl alcohol and methylenecyclopropyl alcohol cannot be considered as suicide substrates, as compared to analogous substrates of other flavin oxidases.     

Mechanism-based inhibitors of dopamine beta-hydroxylase

The copper-containing monooxygenase dopamine beta-hydroxylase catalyzes the hydroxylation of dopamine at the benzylic position to form norepinephrine. Mechanism-based inhibitors for dopamine beta-hydroxylase have been used as probes of the mechanism of catalysis. The variety of such inhibitors that have been developed for this enzyme can be divided into three groups: (i) those in which the inactivating species is formed by abstraction of a hydrogen atom to form a radical intermediate; (ii) those in which the inactivating species is formed by abstraction of an electron to form an epoxide-like intermediate; and (iii) those in which the product is the inactivating species. A mechanism consistent with inactivation by all three groups of inhibitors which proposes that hydroxylation of dopamine by dopamine beta-hydroxylase involves formation of a benzylic radical has been developed. The benzylic radical is formed by abstraction of a hydrogen atom from the substrate by a high-potential copper-oxygen species.     

"Suicide" inactivation of thromboxane A2 synthase. Characteristics of mechanism-based inactivation with isolated enzyme and intact platelets

"Suicide" inactivation occurs during catalysis by thromboxane synthase. Loss of enzymatic activity, accompanying thromboxane B2 formation, was proportional to the substrate concentration. Inactivation was directly related to product formation: for several different experimental protocols 50% loss of thromboxane synthase activity corresponded with formation of 454 +/- 79 ng of thromboxane B2/mg protein. The time course of inactivation was pseudo-first-order and obeyed saturation kinetics. Inactivation (KI) and first-order rate constants (ki) were 18 microM and 0.18 s-1 for prostaglandin H2. Prostaglandin H1, a poor substrate for turnover, was also a site-directed inactivator with KI = 28 microM and ki = 0.09 s-1. Competitive inhibitors, typified by U63557a and U46619, preserved the enzyme activity by slowing the rate of inactivation from 0.18 to 0.05 s-1. Loss of the hemoprotein Soret absorbance did not correlate quantitatively or temporally with the loss of thromboxane synthase activity. A similar, irreversible inactivation accompanied thromboxane formation by intact platelets. Loss of activity was proportional to substrate concentration and catalytic activity. For a pool of 25 separate donors, thromboxane synthase activity declined exponentially as a function of thromboxane B2 formation: 50% loss of activity corresponded to 23 ng of thromboxane B2/10(7) platelets. The data conform to criteria for a specific, mechanism-based process in which thromboxane synthase participates in two parallel reactions, one leading to thromboxane formation and the other to suicide inactivation. The specific, rather than indiscriminate, nature of the process, and its occurrence in intact platelets may have implications for the cell biology of thrombosis. Depletion of thromboxane synthase activity may be a factor in the choice and effectiveness of antithrombotic agents.     

Inactivation of macrophage nitric oxide synthase activity by NG-methyl-L-arginine

.N = O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide (.N = O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of .N = O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by .N = O. NG-Methyl-L-arginine was a competitive inhibitor of .N = O synthase activity derived from activated murine macrophages with a Ki of 6.2 microM. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37 degrees C the kinact was 0.050 min-1. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine acts as a mechanism-based enzyme inactivator of murine macrophage .N = O synthase.     

Chemical modification of dopamine beta-hydroxylase

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.     

Active site labeling of dopamine beta-hydroxylase by two mechanism-based inhibitors: 6-hydroxybenzofuran and phenylhydrazine

6-Hydroxybenzofuran and phenylhydrazine are mechanism-based inhibitors of dopamine beta-hydroxylase (D beta H; EC 1.14.17.1). We report here the isolation and characterization of radiolabeled peptides obtained after inactivation of D beta H with [3H]6-hydroxybenzofuran and [14C]phenylhydrazine followed by digestion with Staphylococcus aureus V8 protease. Inactivation of D beta H with [3H]6-hydroxybenzofuran gave only one labeled peptide, whereas inactivation with [14C]phenylhydrazine gave several labeled peptides. Each inhibitor labeled a unique tyrosine in the enzyme corresponding to Tyr477 in the primary sequence of the bovine enzyme (Robertson, J. G., Desai, P. R., Kumar, A., Farrington, G. K., Fitzpatrick, P. F., and Villafranca, J. J. (1990) J. Biol. Chem. 265, 1029-1035). In addition, [14C]phenylhydrazine also labeled a unique histidine (His249) as well as several other peptides. Examination of the complete peptide profile obtained by high pressure liquid chromatography analysis also revealed the presence of a modified but nonradioactive peptide. This peptide was isolated and sequenced and was identical whether the enzyme was inactivated by 6-hydroxybenzofuran or phenylhydrazine. An arginine at position 503 was missing from the sequence cycle performed by Edman degradation of the modified peptide, but arginine was present in the identical peptide isolated from native dopamine beta-hydroxylase. These data are analyzed based on an inactivation mechanism involving formation of enzyme bound radicals (Fitzpatrick, P. F., and Villafranca, J. J. (1986) J. Biol. Chem. 261, 4510-4518) interacting with active site amino acids that may have a role in substrate binding and binding of the copper ions at the active site.     

Indole protects tryptophan indole-lyase, but not tryptophan synthase, from inactivation by trifluoroalanine.

Trifluoroalanine is a mechanism-based inactivator of Escherichia coli tryptophan indole-lyase (tryptophanase) and E. coli tryptophan synthase (R. B. Silverman and R. H. Abeles, 1976, Biochemistry 15, 4718-4723). We have found that indole is able to prevent inactivation of tryptophan indole-lyase by trifluoroalanine. The protection of tryptophan indole-lyase by indole exhibits saturation kinetics, with a KD of 0.03 mM, which is comparable to the KI for inhibition of pyruvate ion formation (0.01 mM) and the Km for L-tryptophan synthesis. Fluoride electrode measurements indicate the formation of 28 mol of fluoride ion per mole of enzyme during inactivation of tryptophan indole-lyase, and 121 mol of fluoride ion are formed per mole of enzyme in the presence of 2 mM indole during the same incubation period. 19F NMR spectra of reaction mixtures of tryptophan indole-lyase and trifluoroalanine showed evidence only for fluoride ion formation, in either the absence or the presence of indole, and difluoropyruvic acid was not detected. The partition ratio, kcat/kinact, is estimated to be 9. Tryptophan indole-lyase in the presence of trifluoroalanine exhibits visible absorption peaks at 446 and 478 nm, which decay at the same rate as inactivation. However, in the presence of 1 mM indole and trifluoralanine, tryptophan indole-lyase exhibits a peak only at 420 nm, and the spectra show a gradual increase at 300-310 nm with incubation. In contrast, tryptophan synthase is not protected by indole from inactivation by trifluoroalanine, and the absorption peak at 408 nm for the tryptophan synthase-trifluoroalanine complex is unaffected by indole. These results demonstrate that inactivation of tryptophan indole-lyase occurs via a catalytically competent species, probably the beta,beta-difluoro-alpha-aminoacrylate intermediate, which can be partitioned from inactivation to products by a reactive aromatic nucleophile, indole.     

trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.     

Kinetic and spectroscopic studies of the interaction of copper with dopamine beta-hydroxylase

The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.     

A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator.

c[Arg-aB-(CH2+SCH3 phi)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (u-PA and t-PA) and plasmin. This compound inhibited u-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor: first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-1 and 9 microM, respectively at pH 7.5 and 25 degrees C. The activation of plasminogen by u-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than u-PA, respectively.     

Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2'-ethyl- and 3'-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.     

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)     

Selective inactivation by 21-chlorinated steroids of rabbit liver and adrenal microsomal cytochromes P-450 involved in progesterone hydroxylation

The inactivation by 21-chlorinated steroids of rabbit liver cytochromes P-450 involved in the hydroxylation of progesterone has been investigated in intact microsomes encompassing two phenotypes of 21-hydroxylase activity, two phenotypes of 16 alpha-hydroxylase activity, and three phenotypes of 6 beta-hydroxylase activity. In liver microsomes from outbred New Zealand White male rabbits exhibiting a high content of cytochrome P-450 1, 21,21-dichloropregnenolone caused a time- and NADPH-dependent loss of 21-hydroxylase activity. This loss of activity exhibited a number of characteristics of mechanism-based inactivation, including irreversibility, saturation with increasing inhibitor concentrations, and protection by substrate, and was also documented with purified P-450 1 in a reconstituted system. 21,21-Dichloropregnenolone caused no time-dependent loss of 6 beta-hydroxylase activity in microsomes from the New Zealand White rabbits or from control or rifampicin-treated rabbits of the inbred B/J strain. In contrast, in the microsomes from the B/J rabbits, some inactivation of the 16 alpha-hydroxylase was observed (k = 0.04 min-1), regardless of the rifampicin treatment. The other two compounds tested, 21-chloropregnenolone and 21,21-dichloroprogesterone, were less effective than the dichloropregnenolone as inactivators of cytochrome P-450 1. On the other hand, 21,21-dichloroprogesterone, but not 21,21-dichloropregneolone, caused a rapid time-dependent loss of 21-hydroxylase activity in rabbit adrenal microsomes. The results indicate that the introduction of a dichloromethyl group into a substrate bearing a methyl group normally hydroxylated by only one or a few forms of cytochrome P-450 may be a rational means of designing selective inhibitors of the enzyme.