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.     

Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX

Catalytic turnover of sodium azide by horseradish peroxidase, which produces the azidyl radical, results in inactivation of the enzyme with KI = 1.47 mM and kinact = 0.69 min-1. Inactivation of 80% of the enzyme requires approximately 60 equiv each of NaN3 and H2O2. The enzyme is completely inactivated by higher concentrations of these two agents. meso-Azidoheme as well as some residual heme are obtained when the prosthetic group of the partially inactivated enzyme is isolated and characterized. Reconstitution of horseradish peroxidase with meso-azidoheme yields an enzyme without detectable catalytic activity even though reconstitution with heme itself gives fully active enzyme. The finding that catalytically generated nitrogen radicals add to the meso carbon of heme shows that biological meso additions are not restricted to carbon radicals. The analogous addition of oxygen radicals may trigger the normal and/or pathological degradation of heme.     

Synthesis and evaluation of non-steroidal mechanism-based inactivators of 3 alpha-hydroxysteroid dehydrogenase.

Two non-steroidal mechanism-based inactivators for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) of rat liver have been synthesized: 1-(4'-nitrophenyl)-2-propen-1-ol (I), and 1-(4'-nitrophenyl)-2-propyn-1-ol (II). Both of these compounds inactivate homogeneous 3 alpha-HSD in a time- and concentration-dependent manner only in the presence of NAD+. Analysis of the pseudo-first-order inactivation data gave a Kd of 1.2 mM for the allylic alcohol and a t1/2 (time required to promote a 50% loss of enzyme activity) for the enzyme of less than 10 s at saturation. Similar inactivation studies with the acetylenic alcohol gave a Kd of 1.5 mM and a t1/2 for the enzyme of 9.9 min at saturation. The allylic alcohol and acetylenic alcohol are oxidized stereoselectively by the enzyme, yielding a Km of 2.0 mM and a Vmax. of 0.58 mumol/min per mg for the allylic alcohol and a Km of 0.75 mM and a Vmax. of 0.29 mumol/min per mg for the acetylenic alcohol. Effective partition ratios (kcat./kinact.) are low for both alcohols: for the allylic alcohol, 5.3; and for the acetylenic alcohol, 141. H.p.l.c. indicates that the Michael acceptors 1-(4'-nitrophenyl)-2-propen-1-one (III) and 1-(4'-nitrophenyl-2-propyn-1-one (IV) are the products of the enzymic oxidation of the corresponding alcohols. The latter compound (IV) was trapped as its monothioether adducts before h.p.l.c. analysis. The Michael acceptors III and IV inactivate the 3 alpha-HSD in the absence of NAD+ at a rate too high to accurately measure and titrate the enzyme in a stoichiometric manner. Enzyme inactivated by I and NAD+, II and NAD+, III or IV is not re-activated by gel filtration or dialysis, implying a stable covalent bond has been formed between the enzyme and the inactivators. A screen of five other HSDs, and two aliphatic alcohol dehydrogenases, indicates that alcohol I is a selective inactivator of rat liver 3 alpha-HSD. It is concluded that 3 alpha-HSD generates non-steroidal alkylating agents (III and IV) that potently inactivate the enzyme with low effective partition coefficients. This report of non-steroidal mechanism-based inactivators of 3 alpha-HSD may provide a precedent for the development of related compounds to act as suicide substrates of other HSDs.     

Structure and catalytic mechanism of horseradish peroxidase. Regiospecific meso alkylation of the prosthetic heme group by alkylhydrazines

Horseradish peroxidase is inactivated in a time-, H2O2-, and concentration-dependent manner by phenylethyl-, ethyl-, and methylhydrazine. The pseudo- first order kinetic constants for these inactivation reactions at pH 7 are: phenylethyl (KI = 115 microM, kinact = 1.5 min-1, partition ratio = 11), ethyl (KI = 145 microM, kinact = 0.08 min-1, partition ratio = 32), and methyl (KI = 3000 microM, kinact = 0.12 min-1, partition ratio = 80). At pH 5, the constants for the phenylethyl reaction change to KI = 1540 microM and kinact = 0.86 min-1. A transient absorbance at approximately 830 nm, suggestive of an isoporphyrin intermediate, is seen during these reactions. The prosthetic heme is converted by each of the three alkylhydrazines into the corresponding delta-meso-alkylated heme. Complete inactivation of the enzymes by methyl-, ethyl-, and phenylethylhydrazine is associated with alkylation of 60-70, 70, and 90%, respectively, of the prosthetic heme groups. The absence of N-alkylation and the high specificity for the delta-meso position, even with agents as small as methylhydrazine, strengthen the proposal that electron abstraction is mediated by the heme edge rather than the ferryl oxygen of horseradish peroxidase.     

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.     

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.     

Adenine nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(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.     

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)     

Cofactor-directed inactivation by nucleophilic amines of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans.

Phenylhydrazine, semicarbazide, aminoguanidine, hydrazine, and hydroxylamine each irreversibly inactivated methylamine dehydrogenase from Paracoccus denitrificans and caused changes in the absorbance spectrum of the protein-bound tryptophan tryptophylquinone [TTQ] prosthetic group. Different spectral perturbations were observed on reaction with each of these inactivators. In each case a stoichiometry of 2 mol per mol of enzyme (1:1 per cofactor) was required to observe complete modification of the absorbance spectrum. Identical changes were observed in the presence and absence of oxygen. The reactions of hydrazine and hydroxylamine were very rapid, with stoichiometric inactivation occurring in less than 30 s. Inactivation by phenylhydrazine and semicarbazide exhibited apparent bimolecular kinetics and second order rate constants for inactivation, respectively, of 25 min-1 mM-1 and 39 min-1 mM-1. In contrast, inactivation by aminoguanidine exhibited saturation behavior and kinetic parameters of KI = 2.5 mM and kinact = 0.5 min-1 were obtained. Ammonium salts did not inactivate the enzyme, but were reversible competitive inhibitors with respect to methylamine. A Ki of 20 mM was obtained for ammonium chloride. A mechanism for the reactions of these compounds with the TTQ cofactor of methylamine dehydrogenase is proposed, and the relationship of these data to the mechanisms of interaction of these compounds with o-quinones and other quinoproteins which possess TTQ and other quinone cofactors is discussed.     

Inhibition of Na,K-ATPase by a new ATP analog, adenosine-5-N'-(2,4-dinitro-5-fluorophenyl)phosphohydrazide.

A new ATP analog, adenosine-5-N'-(2,4-dinitro-5-fluorophenyl) phosphohydrazide (DNPH-AMP), has been synthesized, which is an irreversible inhibitor of Na,K-ATPase. Interaction of the analog with the enzyme in the presence of K+ is described by the scheme: [formula: see text] and corresponding kinetic constants k3 and Ki are found equal to 2.5 min-1 and 1.6 mM. In the presence of Na+ the time course of enzyme inactivation by DNPH-AMP is a biphasic curve in the semilogarithmic plot. The k3 and Ki values calculated for this case according to Fritzsch [Fritzsch (1985) J. Theor. Biol. 117, 397] are equal to 2.45 min-1 and 2.5 mM, respectively. ATP transforms the K(+)-type of Na,K-ATPase inactivation into the one that takes place in the presence of Na+.     

Purification and properties of cytidine deaminase from escherichia coli

A convenient and efficient procedure for the purification of cytidine deaminase (EC 3.5.4.5) from Escherichia coli is reported. The key step involves adsorption of the enzyme from a crude ammonium sulfate fraction onto a cytidine-containing affinity resin, followed by elution with 0.5 M borate buffer. Subsequent chromatography on DEAE-Sepharose results in an overall 1690-fold purification, yielding enzyme with a specific activity of 118 units/mg. Cytidine deaminase has an apparent molecular weight of 54,000 as determined by gel filtration, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a band at molecular weight 35,000. Cytidine deaminase is inhibited by 5-(chloromercuri)cytidine with kinetic behavior typical of active-site-directed inactivation, with KD = 0.09 mM and kinact = 1.25 min-1. The enzyme is protected against inactivation in the presence of substrate, and the inhibition is reversed with high concentrations of mercaptoethanol. This suggests that inactivation is the result of a mercaptide formation between the mercury and an active-site thiol.     

Inactivation of Pseudomonas iron-superoxide dismutase by hydrogen peroxide

Pseudomonas Fe-superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is inactivated by hydrogen peroxide by a mechanism which exhibits saturation kinetics. The pseudo-first-order rate constant of the inactivation increased with increasing pH, with an inflection point around pH 8.5. Two parameters of the inactivation were measured in the pH range 7.8 to 9.0; the total H2O2 concentration at which the enzyme is half-saturated (K inact) was found to be independent of pH (30 mM) and the maximum rate constant for inactivation (k max) increased progressively with increasing pH, from 3.3 min-1 at pH 7.8 to 21 min-1 at pH 9.0. This evidence suggests the presence of an ionization group (pKa approximately 8.5) which does not participate in the binding of H2O2 but which affects the maximum inactivation rate of the enzyme. The loss of dismutase activity of the Fe-superoxide dismutase is accompanied by a modification of 1.6, 1.1 and 0.9 residues of tryptophan, histidine and cysteine, respectively. Since the amino acid residues of the Cr-substituted enzyme, which has no enzymatic activity, were not modified by H2O2, the active iron of the enzyme is essential for the modification of the amino acid residues.     

The substrate specificity, kinetics, and mechanism of glycerate-3-kinase from spinach leaves

Glycerate-3-kinase (EC 2.7.1.31) from spinach leaves shows absolute specificity for D-glycerate as phosphate acceptor, yielding 3-phosphoglycerate as a product. ATP complexed with either Mg2+ or Mn2+ is the preferred phosphate donor. The enzyme has Km (D-glycerate) = 0.25 mM, Km (Mg-ATP) = 0.21 mM, Vmax = 300 mumol min-1 mg protein-1, and a turnover number = 12,000 X min-1. The equilibrium constant for the reaction is approximately 300 at pH 7.8. Pyrophosphate, 3-phosphoglycerate and ribulose 1,5-bisphosphate are the strongest inhibitors among the phosphorylated and nonphosphorylated metabolites tested; however, their regulatory role in vivo is questioned. Substrate kinetics, as well as product and analog inhibition data, are consistent with a sequential random mechanism. The distinct characteristic of the glycerate kinase-catalyzed reaction is the formation of a dead-end complex between the enzyme, D-glycerate, and 3-phosphoglycerate.     

Structural rearrangements in soluble mitochondrial ATPase

Treatment of isolated factor F1 by 1% dimethylsuberimidate in the presence of 50 mM (NH4)2SO4 leads to the formation of four different types of cross-linked dimers of the subunits, on average one dimer per molecule of the enzyme. This treatment results in 60-70% inactivation of factor F1. Factor F1 treated with dimethylsuberimidate does not show a change in the sedimentation coefficient and is not inactivated in the cold; it is not inactivated in the presence of Mg2+ either, nor is it activated by anions. Incubation of the cross-linked factor F1 with ADP does not lead to inactivation, although the ability to tightly bind ADP is retained. The total quantity of tightly bound ADP reaches 5 mol per mol of the cross-linked factor F1. Cross-linking of factor F1 also prevents the slow inactivation of the enzyme coupled with the hydrolysis of Mg-ATP and Mg-GTP. The dependence of the inactivation rate constant on the concentration of Mg-ATP and Mg-GTP at substrate concentrations of 0.05-2 mM is characterized by the same values of Km,app as those of the ATPase and GTPase activities of factor F1. The probability of the inactivation of factor F1 per turnover remains constant for all the concentrations of the substrates studied and is 2 . 10(-6) per turnover for the ATPase reaction and 2 . 10(-5) per turnover for the GTPase reaction. Moderate hydrostatic pressure (up to 150 atmospheres) greatly accelerates ATP-induced inactivation of factor F1. The activation volume (delta V*) of the inactivation process is equal to 5.1 . 10(-4) cm3/g, which is evidence of considerable changes in the extent of protein hydration during inactivation. Inactivation of the enzyme under pressure is accompanied by dissociation into subunits. Dimethyladipimidate, which does not cause intersubunit cross-linking in the molecule of factor F1, does not alter the properties of the native enzyme. It is suggested that the formation of one intersubunit cross-link in the molecule of factor F1 by dimethylsuberimidate affects the ability of the enzyme to undergo co-operative rearrangements of the quaternary structure under the influence of Mg2+, ADP, ATP, anions, and low temperature. The rate constants of ATP binding to the active site of factor F2 (k+1) = 2 . 10(8) M-1 . min-1), of ATP release from the active site (k-1 = 2 . 10(-2) min-1), and of ADP and Pi release from the active site (k2 = 5 . 10(3) min-1) have been determined. The results obtained confirm the correctness of Boyer's idea, according to which ATP is formed in the active site of mitochondrial ATPase without any external source of energy. Energy is used at the stage of the release of synthesized ATP from the active site of ATPase in the solution.     

Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator

UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5'-diphosphate chloroacetol (UDC) and uridine 5'-diphosphate bromoacetol (UDB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a KD of 0.110 mM and kinact of 0.84 min-1 at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD+. The inactivation of epimerase by uridine 5'-diphosphate [2H2]chloroacetol proceeds with a primary kinetic isotope effect (kH/kD) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD+ at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD+ is proposed to be the chromophore with lambda max at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.     

Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures.

1. Alcohol oxidase (alcohol: oxygen oxidoreductase) of a thermophilic methanol-utilizing yeast, Hansenula polymorpha DL-1, was isolated in crystalline form. 2. This alcohol oxidase of H. polymorpha was more stable to heat than was the enzyme of Kloeckera sp. This difference in heat stability is compatible with the difference in growth temperatures for both yeasts. 3. The crystalline alcohol oxidases of both yeast oxidized the lower primary alcohols (C-2 to C-4) as well as methanol. The apparent Km values for the methanol of Kloeckera and H. polymorpha enzymes were 0.44 and 0.23 mM, respectively. The enzymes could also oxidize formaldehyde to formate, and were inactivated by relatively low concentrations of hydrogen peroxide. 4. The molecular weight for both enzymes was calculated to be about 670000. Each enzyme is composed of eight identical subunits (molecular weight 83000) and contains eight moles of FAD as the prosthetic group. The NH2-terminal and COOH-terminal amino acids of H. polymorpha enzyme were identified as alanine and phenylalanine, respectively. The octameric subunits model of each enzyme was confirmed by electron micrographs, which showed an octad aggregate, composed of two tetragons face to face.     

Inactivation of Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase by an enzyme-generated ethoxyacetylenic ketone in the presence of a thiol

Replacement of the 21-methyl group of 20 beta-hydroxypregn-4-en-3-one with an ethoxyacetylene group yields a compound that is an excellent substrate (pH 7.4, Km = 2.3 microM, Vmax = 4.6 nmol min-1 micrograms-1) for the Streptomyces hydrogenans NAD(H)-dependent 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53). The enzyme-generated ethoxyacetylenic ketone product is a potent inactivator of the enzyme. Gel filtration chromatography of enzyme inactivated with radiolabeled steroid demonstrates that covalent modification of the enzyme has occurred. Both NAD and NADH retard the rate of inactivation, suggesting that only free enzyme is susceptible to covalent modification. Consequently, enzymatically formed ethoxyacetylenic ketone does not react with the enzyme while it is part of the ternary complex. Moreover, the kinetically preferred release of this reactive ketone prior to NADH release assures that enzyme inactivation occurs only when released ketone subsequently encounters free enzyme. Kinetic analysis of inactivations carried out with chemically prepared ethoxyacetylenic ketone and enzyme at pH 7.4 and 9.2 yields bimolecular rate constants for the inactivation process of 1.15 X 10(4) L mol-1 s-1 and 6.94 X 10(4) L mol-1 s-1, respectively. This bimolecular reaction is faster than the bimolecular reaction of the ethoxyacetylenic ketone with either glutathione, mercaptoethanol, or dithiothreitol. Thus, complete inactivation by ketone generated from 5 microM alcohol and 5 microM NAD occurs in 30 min at pH 7.4 in the presence of 1 mM glutathione.     

The involvement of catalase in alcohol metabolism in Drosophila melanogaster larvae

The involvement of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) in the metabolism of alcohols was investigated by comparing Drosophila melanogaster larvae in which catalase was inhibited by dietary 3-amino-1,2,4-triazole (3AT) to larvae fed a diet without 3AT. 3AT inhibited up to 80% of the catalase activity with concordant small increases in the in vitro activities of sn-glycerol-3-phosphate dehydrogenase, fumarase, and malic enzyme, but with a 16% reduction in the in vivo incorporation of label from [14C]glucose into lipid. When the catalase activity was inhibited to different degrees in ADH-null larvae, there was a simple linear correlation between the catalase activity and flux from [14C]ethanol into lipid. By feeding alcohols simultaneously with 3AT, ethanol and methanol were shown to react efficiently with catalase in wild-type larvae at moderately low dietary concentrations. Drosophila catalase did not react with other longer chain alcohols. Catalase apparently represents a minor pathway for ethanol degradation in D. melanogaster larvae, but it may be an important route for methanol elimination from D. melanogaster larvae.     

Biosynthetic thiolase from Zoogloea ramigera. Evidence for a mechanism involving Cys-378 as the active site base.

Biosynthetic thiolase from Zoogloea ramigera was inactivated with a mechanism-based inactivator, 3-pentynoyl-S-pantetheine-11-pivalate (3-pentynoyl-SPP) where K1 = 1.25 mM and kinact = 0.26 min-1, 2,3-pentadienoyl-SPP obtained from nonenzymatic rearrangement of 3-pentynoyl-SPP where K1 = 1.54 mM and kinact = 1.9 min-1 and an affinity labeling reagent, acryl-SPP. The results obtained with the alkynoyl and allenoyl inactivators are taken as evidence that thiolase from Z. ramigera is able to catalyze proton abstraction uncoupled from carbon-carbon bond formation. The inactivator, 3-pentynoyl-SPP and the affinity labeling reagent, acryl-SPP, trap the same active site cysteine residue, Cys-378. To assess if Cys-378 is the active site residue involved in deprotonation of the second molecule of acetyl-CoA, a Gly-378 mutant enzyme was studied. In the thiolysis direction the Gly-378 mutant was more than 50,000-fold slower than wild type and over 100,000-fold slower in the condensation direction. However, the mutant enzyme was still capable of forming the acetyl-enzyme intermediate and incorporated 0.81 equivalents of 14C-label after incubation with [14C]Ac-CoA for 60 min. The reversible exchange of 32P-label from [32P]CoASH into Ac-CoA, catalyzed by the Gly-378 mutant enzyme, proceeded with a Vmax (exchange) 8,000-fold less than the wild type enzyme but at least 10-fold faster than the overall condensation reaction. These data provide evidence that Cys-378 is the active site base.