Stereoselectivity and regioselectivity of uridine-5'-diphosphoglucuronosyltransferase toward vicinal dihydrodiols of polycyclic aromatic hydrocarbons

The ability of a purified rat liver microsomal uridine-5'-diphosphoglucuronosyltransferase to catalyze the glucuronidation of stereoisomeric trans- and cis-9, 10-dihydroxy-9, 10-dihydrophenanthrenes and 4, 5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes is examined. The enzyme shows the ability to discriminate kinetically between the antipodes of trans-9, 10-dihydroxy-9, 10-dihydrophenanthrene with turnover numbers of 0.070 and 1.4 s-1 and kc/Kmapp values of 4.4 X 10(3) and 1.1 X 10(3) M-1 s-1 for the 9R, 10R and 9S, 10S stereoisomers. Glucuronidation of the nondissymmetric cis-9, 10-dihydroxy-9, 10-dihydrophenanthrene proceeds with a turnover number of 0.037 s-1 and kc/Kmapp of 18 X 10(3) M-1 s-1 to give a 60/40 mixture of the two possible diastereomeric products. Three of the four stereoisomers of 4,5-dihydroxy-4,5-dihydrobenzo[alpha] pyrene are regioselectively glucuronidated by the enzyme with a high degree of kinetic discrimination. Turnover numbers for the 4S,5S, 4R,5R, and 4S,5R stereoisomers are 4.1, 0.37, and 0.23 s-1 with kc/Kmapp values of 23.8 X 10(3), 0.23 X 10(3), and 3.15 X 10(3) M-1 s-1, respectively. The 4R,5S cis isomer is not a substrate. Enzyme-catalyzed reactions of the 4S,5S and 4S,5R isomers give exclusively (greater than or equal to 95%) the 4-glucuronide with the 4R,5R isomer giving the 5-glucuronide. The kinetic and regiochemical results indicate that the enzyme recognizes hydroxyl groups on the beta-face or bottom face of the 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutathione depletion and cytotoxicity by naphthalene 1,2-oxide in isolated hepatocytes

The ability of naphthalene 1,2-oxide to diffuse across intact cellular membranes, the subsequent biotransformation of this epoxide and its potential to produce losses in cellular viability have been examined in incubations of isolated hepatocytes. Addition of 1R,2S- or 1S,2R-naphthalene oxide enantiomers (15, 30 and 60 microM) to isolated hepatocytes resulted in a rapid depletion of intracellular glutathione. Depletion of glutathione was concentration dependent and maximal at 5-15 min. Addition of either of the enantiomeric oxides at 60 microM resulted in the loss of more than 20 nmol glutathione/10(6) cells (1 ml cells); thus more than a third of the added epoxide was available for conjugation with intracellular glutathione. The time course and concentration dependence of glutathione depletion corresponded to the rapid, concentration-dependent formation of naphthalene oxide glutathione conjugates. The levels of glutathione adduct were highest 1 min after addition of naphthalene oxide and declined to 25% of this level after 30 min. Loss of glutathione conjugates from incubations correlated with the formation of N-acetylcysteine adducts. In contrast, the levels of glutathione adducts added exogenously to hepatocytes were relatively stable over a 120-min incubation suggesting that although further metabolism of naphthalene oxide glutathione adducts formed intracellularly is possible, extracellular glutathione adducts cannot penetrate the hepatocellular membrane. Small amounts of radiolabel from [3H]naphthalene 1,2-oxide were bound covalently to macromolecules in hepatocytes; the rate of this binding slowed rapidly after the first minute of incubation. Severe blebbing of the surface of the hepatocytes was noted in cells incubated for 30 min with 480 microM naphthalene oxide. Many of the cells were vacuolated at 60 min and progressed to frank necrosis with pyknotic nuclei and inability to exclude trypan blue. Cells incubated with 1-naphthol responded in a qualitatively similar fashion to those cells incubated with epoxide; however, hepatocytes incubated with 1-naphthol progressed to frank cellular necrosis at a slower rate. In hepatocytes partially depleted of glutathione by pretreatment with buthionine sulfoximine, addition of 1S,2R-naphthalene oxide at a rate of 1 nmol/min/10(6) cells resulted in significant losses in cell viability. In contrast, no losses in cell viability were observed with the enantiomer, 1R,2S-naphthalene oxide. Both epoxides produced similar losses in cellular glutathione levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition.

The O2-independent hydroxylase 4-ethylphenol methylenehydroxylase (4EPMH) from Pseudomonas putida JD1 catalysed the complete conversion of 4-ethylphenol into 1-(4-hydroxyphenyl)ethanol together with a small amount of 4-hydroxyacetophenone, but with no formation of the side product 4-vinylphenol reported to be formed when the similar enzyme p-cresol methylhydroxylase (PCMH) catalyses this reaction. The enantiomer of 1-(4-hydroxyphenyl)ethanol produced by 4EPMH was R(+) when horse heart cytochrome c or azurin was used as electron acceptor for the enzyme. PCMHs from various bacterial strains produced the S(-)-alcohol. Both enantiomers of 1-(4-hydroxyphenyl)ethanol were substrates for conversion into 4-hydroxyacetophenone by 4EPMH, but the S(-)-isomer was preferred. The Km and kcat. were 1.2 mM and 41 s-1 respectively for the S(-)-alcohol and 4.7 mM and 22 s-1 for the R(+)-alcohol. In addition to the 1-(4-hydroxyphenyl)ethanol dehydrogenase activity of 4-EPMH, NAD(+)-linked dehydrogenase activity for both enantiomers of the alcohol was found in extracts of Ps. putida JD1.     

Some redox properties of myohemerythrin from retractor muscle of Themiste zostericola

Distinct semimetmyohemerythrin species are produced by one-electron oxidation of deoxymyohemerythrin and one-electron reduction of metmyohemerythrin. The former, (semimetmyo)o, changes (greater than or equal to 90%) to the latter, (semimetmyo)R, with k = 1.0 x 10(-2) s-1, delta H = 15.1 kcal mol-1 and delta S = -17 eu. Oxidation of (semimetmyo)o by Fe(CN)6(3)- rapidly produces an unstable metmyohemerythrin form which converts to the final metmyohemerythrin with k = 4.6 x 10(-3) s-1, delta H = 16.8 kcal mol-1, and delta S = -13 eu. The two met forms react at the same rate with N3-, but the unstable form reacts very rapidly with S2O4(2-) in contrast to stable metmyohemerythrin. (Semimetmyo)R or a mixture of metmyohemerythrin and deoxymyohemerythrin equilibrate very slowly to a mixture containing all three species. The rate constants for disproportionation and comproportionation are 0.89 M-1 s-1 and 9.4 M-1 s-1, respectively. EPR spectra near liquid He temperatures and optical absorption spectra have been used to characterize and measure the rates at 25 degrees C, pH 8.2, and I = 0.15 M. The comparative behavior of octameric and monomeric protein is discussed.     

The chlorinating activity of human myeloperoxidase: high initial activity at neutral pH value and activation by electron donors

The steady-state activity of myeloperoxidase in the chlorination of monochlorodimedone at neutral pH was investigated. Using a stopped-flow spectrophotometer we were able to show that the enzymic activity at pH 7.2 rapidly declined in time. During the first 50-100 ms after addition of H2O2 to the enzyme, a turnover number of about 320 s-1 per haem was observed. However, this activity decreased rapidly to a value of about 25s-1 after 1 s. This shows that in classical steady-state activity measurements, the real activity of the enzyme at neutral pH is grossly underestimated. By following the transient spectra of myeloperoxidase during turnover it was shown that the decrease in activity was probably caused by the formation of an enzymically inactive form of the enzyme, Compound II. As demonstrated before (Bolscher, B.G.J.M., Zoutberg, G.R., Cuperus, R.A. and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191) reductants such as ascorbic acid and ferrocyanide convert Compound II, which accumulates during turnover, into active myeloperoxidase. Activity measurements in the presence of ascorbic acid showed, indeed, that the moderate enzymic activity was higher than in the absence of ascorbic acid. With 5-aminosalicylic acid present, however, the myeloperoxidase activity remained at a much higher level, namely about 150 s-1 per haem during the time interval from 100 ms to 5 s after mixing. From combined stopped-flow/rapid-scan experiments during turnover it became clear that in the presence of 5-aminosalicylic acid the initially formed Compound II was rapidly converted back to native enzyme. Presteady-state experiments showed that 5-aminosalicylic acid reacted with Compound II with a K2 of 3.2 x 10(5) M-1.s-1, whereas for ascorbic acid a K2 of 1.5 x 10(4) M-1.s-1 was measured at pH 7.2. In the presence of 5-aminosalicylic acid during the time interval in which the myeloperoxidase activity remained constant, a Km for H2O2 at pH 7.2 was determined of about 30 microM at 200 mM chloride. In the absence of reductants the same value was found during the first 100 ms after addition of H2O2 to the enzyme. The physiological consequences of these findings are discussed.     

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.     

Optimization of efficiency in the glyoxalase pathway

A quantitative kinetic model for the glutathione-dependent conversion of methylglyoxal to D-lactate in mammalian erythrocytes has been formulated, on the basis of the measured or calculated rate and equilibrium constants associated with (a) the hydration of methylglyoxal, (b) the specific base catalyzed formation of glutathione-(R,S)-methylglyoxal thiohemiacetals, (c) the glyoxalase I catalyzed conversion of the diastereotopic thiohemiacetals to (S)-D-lactoylglutathione, and (d) the glyoxalase II catalyzed hydrolysis of (S)-D-lactoylglutathione to form D-lactate and glutathione. The model exhibits the following properties under conditions where substrate concentrations are small in comparison to the Km values for the glyoxalase enzymes: The overall rate of conversion of methylglyoxal to D-lactate is primarily limited by the rate of formation of the diastereotopic thiohemiacetals. The hydration of methylglyoxal is kinetically unimportant, since the apparent rate constant for hydration is (approximately 500-10(3))-fold smaller than that for formation of the thiohemiacetals. The rate of conversion of methylglyoxal to (S)-D-lactoylglutathione is near optimal, on the basis that the apparent rate constant for the glyoxalase I reaction (kcatEt/Km congruent to 4-20 s-1 for pig, rat, and human erythrocytes) is roughly equal to the apparent rate constant for decomposition of the thiohemiacetals to form glutathione and methylglyoxal [k(obsd) = 11 s-1, pH 7]. The capacity of glyoxalase I to use both diastereotopic thiohemiacetals, versus only one of the diastereomers, as substrates represents a 3- to 6-fold advantage in the steady-state rate of conversion of the diastereomers to (S)-D-lactoylglutathione.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissection of the catalytic mechanism of isozyme 4-4 of glutathione S-transferase with alternative substrates

The kinetic and chemical mechanism of isozyme 4-4 of rat liver glutathione (GSH) S-transferase was investigated by using several alternative peptide substrates including N-acetyl-GSH, gamma-L-glutamyl-L-cysteine (gamma-GluCys), N4-(malonyl-D-cysteinyl)-L-2,4-diaminobutyrate (retro-GSH), and N4-(N-acetyl-D-cysteinyl)-L-2,4-diaminobutyrate (decarboxylated retro-GSH). The enzyme, which is normally stereospecific in the addition of GSH to the oxirane carbon of R absolute configuration in arene oxide substrates, loses its stereospecificity toward phenanthrene 9,10-oxide with the retro peptide analogues, giving a 2:1 mixture of the S,S and R,R stereoisomeric 9,10-dihydro-9-(S-peptidyl)-10-hydroxyphenanthrenes. The analogues with normal peptide bonds (N-acetyl-GSH and gamma-GluCys) show normal stereospecific addition. The kinetic mechanism of the enzyme was investigated by using the alternative substrate diagnostic with several 4-substituted 1-chloro-2-nitrobenzenes and GSH, N-acetyl-GSH, and gamma-GluCys. Varying the concentration of electrophile vs the identity of the GSH analogue and the concentration of GSH vs the identity of the electrophile gave two sets of intersecting reciprocal plots, a result consistent with a random sequential kinetic mechanism. The pH profiles of kc and kc/Ksm [saturating GSH, variable 1-chloro-2,4-dinitrobenzene (1)] exhibit a dependence on a deprotonation in the enzyme-GSH-1 and enzyme-GSH complexes with molecular pKa's of 6.1 and 6.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chiral instability at sulfur of S-adenosylmethionine

S-Adenosylmethionine, generated enzymically in chirally pure form (S configuration at sulfur), undergoes simultaneous irreversible conversion to 5'-deoxy-5'-(methylthio)adenosine and homoserine with a rate constant of 6 X 10(-6) s-1 at pH 7.5 and 37 degrees C and reversible conversion to an enzymically inactive stereoisomer (R configuration at sulfur) with a forward rate constant of 8 X 10(-6) s-1 at pH 7.5 and 37 degrees C. These forms of instability require small turnover times and/or stabilization through macromolecular binding for S-adenosylmethionine, if organisms that utilize it are to avoid losses of metabolic energy.     

Reactions of the Wurster's red radical cation with hemoglobin and glutathione during the cooxidation of N,N-dimethyl-p-phenylenediamine [correction of phenlenediamine] and oxyhemoglobin in human red cells.

N,N-Dimethyl-p-phenylenediamine (DMPD) reacted directly with oxyhemoglobin under formation of ferrihemoglobin and, presumably, the N,N-dimethyl-p-phenylenediamine radical cation (DMPP.+). The apparent second-order rate constant of this reaction was 1 M-1 s-1 (pH 7.4, 37 degrees C). The reaction rate was diminished by catalase (by 1/3) and by superoxide dismutase (by 1/5). The apparent second-order rate constant of ferrihemoglobin formation by DMPD.+ was 5 x 10(3) M-1 s-1. Since DMPD.+ is disproportionated by 50% at pH 7.4, the quinonediimine could not be excluded as the ultimate ferrihemoglobin forming oxidant. To prove this hypothesis, the disproportionation equilibrium was shifted to the radical side by addition of excess DMPD. Ferrihemoglobin formation was thereby increased, indication that the radical was the responsible oxidant. In contrast to ferrihemoglobin formation, reactions with glutathione occurred predominantly with the quinonediimine. The second-order rate constant of this reaction was 4 x 10(5) M-1 s-1 which approaches the value obtained with p-benzoquinone. In contrast to the corresponding reactions of the N,N,N',N'-tetramethyl-p-phenylenediamine radical cation, the disporportionation reaction of DMPD.+ was very fast, k = 2 x 10(6) M-1 s-1. Formation of glutathione disulfide was negligible and the main reaction products were two isomeric glutathione adducts, 2- and 3-(glutathione-S-yl)-N,N-dimethyl-p-phenylenediamine. In human erythrocytes, DMPD produced many equivalents of ferrihemoglobin, diminished glutathione and produced both thioethers. In contrast to ferrihemoglobin formation, DMPD and glutathione disappearance as well as thioether appearance occured only after a marked lag phase. The calculated steady state concentration of DMPD.+ was only 4 x 10(-6) the DMPD concentration, as long as ferrihemoglobin was low. At increasing ferrihemoglobin higher steady state concentrations of the radical are attained. In fact, preformed ferrihemoglobin in red cells significantly accelerated DMPD and glutathione disappearance. This effect was completely prevented in the presence of ferrihemoglobin-complexing cyanide. The presented experiments once more appoint blood as a metabolically competent organ for the biotransformation of aromatic amines.     

Characterization of a variant rat glutathione S-transferase by cDNA expression in Escherichia coli

We have isolated a glutathione S-transferase Yb1 subunit cDNA from a lambda gt11 cDNA collection constructed from rat testis poly(A) RNA enriched for glutathione S-transferase mRNA activities. This Yb1 cDNA, designated pGTR201, is identical to our liver Yb1 cDNA clone pGTR200 except for a shorter 5'-untranslated sequence. Active glutathione S-transferase is expressed from this Yb1 cDNA driven by the tac promoter on the plasmid construct pGTR201-KK. The expressed glutathione S-transferase protein begins with the third codon (Met) of the cDNA, and is missing the N-terminal proline of rat liver glutathione S-transferase 3-3. Therefore, our Escherichia coli expressed glutathione S-transferase protein represents a variant form of glutathione S-transferase 3-3 (Yb1Yb1), designated GST 3-3(-1). The expressed Yb1 subunits are assembled into a dimer as purified from sonicated E. coli crude extracts. In the absence of dithiothreitol three active isomers can be resolved by ion-exchange chromatography. The pure protein has an extinction coefficient of 9.21 x 10(4) M-1 cm-1 at 280 nm or E0.1% 280 = 1.78 and a pI at 8.65. It has a substrate specificity pattern similar to that of the authentic glutathione S-transferase 3-3. The GST 3-3(-1) has a KM of 202 microM for reduced GSH and of 36 microM for 1-chloro-2,4-dinitrobenzene. The turnover number for this conjugation reaction is 57 s-1. Results of kinetic studies of this reaction with GST 3-3(-1) are consistent with a sequential substrate binding mechanism. We conclude that the first amino acid proline of glutathione S-transferase 3-3 is not essential for enzyme activities.     

On the reaction of molecular oxygen with thiyl radicals: a re-examination

Absolute rate constants for the addition of oxygen to thiyl radicals, i.e. RS. + O2----RSOO., have been determined by applying a new competition method based on RS. formation via one-electron reduction of the corresponding disulphides, and the competition between RS. reacting with O2 and an electron donor such as ascorbate. Bimolecular rate constants have been obtained for the thiyl radicals derived from cysteine (6.1 X 10(7) mol-1 dm3 s-1), penicillamine (2.5 X 10(7) mol-1 dm3 s-1), homocysteine (8.0 X 10(7) mol-1 dm3 s-1), cysteamine (2.8 X 10(7) mol-1 dm3 s-1), 3-thiopropionic acid (2.2 X 10(8) mol-1 dm3 s-1) and glutathione (3.0 X 10(7) mol-1 dm3 s-1), respectively. The values obtained for the O2 addition to the thiyl radicals from glutathione and cysteine are considerable lower (by about two orders of magnitude) than those previously published. This indicates that the RS. + O2 reaction may be of complex nature and is generally a process which is not solely controlled by the diffusion of the reactants.     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

Activation/inactivation and uni-site catalysis by the reconstituted ATP-synthase from chloroplasts

The proton-translocating ATP-synthase of chloroplasts, CF0F1, was isolated and reconstituted into asolectin liposomes. CF0F1 can exist in at least four different states, oxidized or reduced, either inactive or active. These states are characterized by different kinetics of ADP binding: There is no binding of ADP to the inactive, oxidized state, the rate constant for ADP binding to the inactive, reduced states is 7.10(2) M-1.s-1. ADP binding to the active, reduced state occurs under deenergized conditions with 10(5) M-1.s-1 and transforms the enzyme into the inactive, reduced state. Parallel to the ADP-dependent inactivation, the enzyme can also inactivate without ADP binding with a first-order rate constant of 7.10(-3) M-1.s-1. With the active, reduced enzyme ATP-hydrolysis was measured under uni-site conditions as has been carried out with MF1 (Grubmeyer, C., Cross, R.C. and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100). The rate constant for ATP binding is 10(6) M-1.s-1, the 'equilibrium constant' on the enzyme EADPPi/EATP is 0.4. The rate constants for Pi release and ADP release are 0.2 s-1 and o.1 s-1, respectively. This indicates that the enzyme carries out a complete turnover under uni-site conditions with rates much higher than that reported for MF1.     

Kinetics of dissociation of reduced nicotinamide adenine dinucleotide phosphate from its complexes with malic enzyme in relation to substrate inhibition and half-of-the-sites reactivity

Malic enzyme of pigeon liver binds NADPH at four equivalent enzyme sites and binds Mn2+ and malate each at two sets of "tight" and "weak" sites with negative cooperativity [Pry, T. A., & Hsu, R. Y. (1980) Biochemistry 19, 951-962]. Stopped-flow studies on the displacement of NADPH from the malate-enzyme complexes E4-NADPH4, E4-Mn2(2+)-NADPH4, E4-Mn2(2+)-NADPH4-dimalate, and E4-Mn2(2+)-NADPH4-tetramalate by large excess NADP+ or its analogue phosphoadenosine(2')diphospho(5')ribose show that NADPH dissociates from the binary complex rapidly with a first-order rate constant of 427 s-1. Dissociation from the ternary E4-Mn2(2+)-NADPH4 complex containing two tightly bound Mn2+ ions can be described by a single first-order process with a rate constant of 135 s-1, or more satisfactorily by two simultaneous first-order processes attributable to the reactions of Mn2+-deficient (k congruent to 427 s-1) and Mn2+-liganded (k = 96 s-1) subunits. The latter equals twice the maximum steady-state turnover rate of 53.2 + 3.0 s-1 assigned to dissociation of the reduced nucleotide from transient E-Mn2+-NADPH, and this 2:1 ratio strongly supports our proposed "half-of-the-sites" model [Hsu, R. Y., & Pry, T. A. (1980) Biochemistry 19, 962-968]. Dissociation from the E4-Mn2(2+)-NADPH4-dimalate complex (k = 100 s-1) follows only the slower process, suggesting that occupancy of malate at two sites tightens enzyme-bound NADPH on the adjacent sites. Binding of malate at two additional weak sites yields E4-Mn2(2+)-NADPH4-tetramalate and a NADPH dissociation rate constant of 2.69 s-1. The 97% decrease in NADPH dissociation parallels the observed 93% maximal inhibition by malate and is the cause of substrate inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

The formation of styrene glutathione adducts catalyzed by prostaglandin H synthase. A possible new mechanism for the formation of glutathione conjugates

The metabolism of styrene by prostaglandin hydroperoxidase and horseradish peroxidase was examined. Ram seminal vesicle microsomes in the presence of arachidonic acid or hydrogen peroxide and glutathione converted styrene to glutathione adducts. Neither styrene 7,8-oxide nor styrene glycol was detected as a product in the incubation. Also, the addition of styrene 7,8-oxide and glutathione to ram seminal vesicle microsomes did not yield styrene glutathione adducts. The peroxidase-generated styrene glutathione adducts were isolated by high pressure liquid chromatography and characterized by NMR and tandem mass spectrometry as a mixture of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione. (1R)- and (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione were not formed by the peroxidase system. The addition of phenol or aminopyrine to incubations, which greatly enhances the oxidation of glutathione to a thiyl radical by peroxidases, increased the formation of styrene glutathione adducts. We propose a new mechanism for the formation of glutathione adducts that is independent of epoxide formation but dependent on the initial oxidation of glutathione to a thiyl radical by the peroxidase, and the subsequent reaction of the thiyl radical with a suitable substrate, such as styrene.     

Peroxidase catalysed aerobic degradation of 5,6,7,8-tetrahydrobiopterin at physiological pH

The aerobic degradation of 5,6,7,8-tetrahydrobiopterin at neutral pH is catalysed by peroxidase (EC 1.11.1.7) and provides quinonoid 7,8-dihydro(6H)biopterin which readily loses the side chain to yield 7,8-dihydro(3H)pterin. The latter is in equilibrium with trace amounts of 6-hydroxy-5,6,7,8-tetrahydropterin (covalent hydrate) which is irreversibly oxidised to quinonoid 6-hydroxy-7,8-dihydro(6H)pterin, and this finally rearranges to 7,8-dihydroxanthopterin. Spectroscopic evidence (ultraviolet, 1H NMR and 13C NMR) is presented for the reversible addition of water across the 5,6-double bond of 7,8-dihydro(3H)pterin. The intermediate quinonoid 6-hydroxy-7,8-dihydro(6H)pterin is a good substrate for dihydropteridine reductase (EC 1.6.99.7) with a Km of 16.3 microM and kcat of 22.5 s-1. The rate of aerobic degradation (oxidation and loss of the side chain) of natural (6R)-5,6,7,8-tetrahydrobiopterin is several times slower than the rate for the unnatural (6S) isomer. By using a modified assay procedure the kinetic parameters for dihydropteridine reductase are as follows: with (6R)-7,8-dihydro(6H)biopterin Km = 1.3 microM and kcat = 22.8 s-1; with (6S)-7,8-dihydro(6H)biopterin Km = 13.5 microM and kcat = 51.6 s-1; and with (6RS)-7,8-dihydro(6H)neopterin Km = 19.2 microM and kcat = 116 s-1.     

One- and two-electron reduction of quinones by glutathione reductase

Yeast glutathione reductase (E.C. 1.6.4.2) catalyzes the oxidation of NADPH by p-quinones and ferricyanide with a maximal turnover number (TNmax) of 4-5 s-1.NADP+ stimulates the reaction and the TNmax/Km value of acceptors is reached at NADP+/NADPH greater than or equal to 100. TNmax is increased up to 30-33 s-1. The stimulatory effect of NADP+ may be associated with its complexation with the NADPH-binding site in the reduced enzyme (Kd = 40-60 microM). It is suggested that NADP+ shifts the electron density towards FAD in the two-electron-reduced enzyme and, evidently, changes its one-electron-reduction potentials, while quinones oxidize an equilibrium form of glutathione reductase containing reduced FAD. In the absence of NADP+ the reduction of quinones by glutathione reductase proceeds mainly in a two-electron manner. At NADP+/NADPH = 100 a one-electron reduction makes up 44% of the total process. At pH 6.0-7.0 the reduced forms of naphthoquinones undergo cyclic redox conversions. A hyperbolic dependence exists of the log TN/Km of quinones on their one-electron-reduction potentials.     

Pre-steady-state and steady-state kinetic analysis of E. coli class I ribonucleotide reductase

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs and is composed of two homodimeric subunits: R1 and R2. R1 binds NDPs and contains binding sites for allosteric effectors that control substrate specificity and turnover rate. R2 contains a diiron-tyrosyl radical (Y(*)) cofactor that initiates nucleotide reduction. Pre-steady-state experiments with wild type R1 or C754S/C759S-R1 and R2 were carried out to determine which step(s) are rate-limiting and whether both active sites of R1 can catalyze nucleotide reduction. Rapid chemical quench experiments monitoring dCDP formation gave k(obs) of 9 +/- 4 s(-1) with an amplitude of 1.7 +/- 0.4 equiv. This amplitude, generated in experiments with pre-reduced R1 (3 or 15 microM) in the absence of reductant, indicates that both monomers of R1 are active. Stopped-flow UV-vis spectroscopy monitoring the concentration of the Y(*) failed to reveal any changes from 2 ms to seconds under similar conditions. These pre-steady-state experiments, in conjunction with the steady-state turnover numbers for dCDP formation of 2-14 s(-1) at RNR concentrations of 0.05-0.4 microM (typical assay conditions), reveal that the rate-determining step is a physical step prior to rapid nucleotide reduction and rapid tyrosine reoxidation to Y(*). Steady-state experiments conducted at RNR concentrations of 3 and 15 microM, typical of pre-steady-state conditions, suggest that, in addition to the slow conformational change(s) prior to chemistry, re-reduction of the active site disulfide to dithiol or a conformational change accompanying this process can also be rate-limiting.     

Catalytic mechanism of dichloromethane dehalogenase from Methylophilus sp. strain DM11

The glutathione (GSH)-dependent dichloromethane dehalogenase from Methylophilus sp. strain DM11 catalyzes the dechlorination of CH(2)Cl(2) to formaldehyde via a highly reactive, genotoxic intermediate, S-(chloromethyl)glutathione (GS-CH(2)Cl). The catalytic mechanism of the enzyme toward a series of dihalomethane and monohaloethane substrates suggests that the initial addition of GSH to the alkylhalides is fast and that the rate-limiting step in turnover is the release of either the peptide product or formaldehyde. With the exception of CH(2)ClF, which forms a relatively stable GS-CH(2)F intermediate, the turnover numbers for a series of dihalomethanes fall in a very narrow range (1-3 s(-1)). The pre-steady-state kinetics of the DM11-catalyzed addition of GSH to CH(3)CH(2)Br exhibits a burst of S-(ethyl)-glutathione (k(b) = 96 +/- 56 s(-1)) followed by a steady state with k(cat) = 0.13 +/- 0.01 s(-1). The turnover numbers for CH(3)CH(2)Cl, CH(3)CH(2)Br, and CH(3)CH(2)I are identical, indicating a common rate-limiting step. The turnover numbers of the enzyme with CH(3)CH(2)Br and CH(3)CH(2)I are dependent on viscosity and are very close to the measured off-rate of GSEt. The turnover number with CH(2)I(2) is also dependent on viscosity, suggesting that a diffusive step is rate-limiting with dihaloalkanes as well. The rate constants for solvolysis of CH(3)SCH(2)Cl, a model for GS-CH(2)Cl, range between 1 s(-1) (1:1 dioxane/water) and 64 s(-1) (1:10 dioxane/water). Solvolysis of the S-(halomethyl)glutathione intermediates may also occur in the active site of the enzyme preventing the release of the genotoxic species. Together, the results indicate that dissociation of the GS-CH(2)X or GS-CH(2)OH intermediates from the enzyme may be a relatively rare event.