Mechanism-based inactivation of 17 beta,20 alpha-hydroxysteroid dehydrogenase by an acetylenic secoestradiol

14,15-Secoestra-1,3,5(10)-trien-15-yne-3,17 beta-diol (1) is a mechanism-based inactivator of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (estradiol dehydrogenase, EC 1.1.1.62). Inactivation with alcohol 1 requires NAD-dependent enzymic oxidation and follows approximately pseudo-first-order kinetics with a limiting t1/2 of 82 min and a "Ki" of 2.0 microM at pH 9.2 and 25 degrees C. At saturating concentrations of NAD, the initial rate of inactivation is slower than in the presence of 5 microM NAD, suggesting that cofactor binding to free enzyme impedes the inactivation process. Glutathione completely protects the enzyme from inactivation at both cofactor concentrations. Inactivation with 45 microM tritiated alcohol 1 followed by dialysis and gel filtration demonstrates a covalent interaction and affords an estimated stoichiometry of 1.4 molecules of steroid per subunit (2.8 per dimer). Chemically prepared 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one (2) rapidly inactivates estradiol dehydrogenase with biphasic kinetics. From the latter phase, a Ki of 2.8 microM and a limiting t1/2 of 12 min at pH 9.2 were determined. Estradiol, NADH, and NAD all retard this latter inactivation phase. We propose that enzymatically generated ketone 2 inactivates estradiol dehydrogenase after its release from and return to the active site of free enzyme.     

Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue

Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acceleration of the NAD cyanide adduct reaction by lactate dehydrogenase: the equilibrium binding effect as a measure of the activation of bound NAD

The binary complex of NAD and lactate dehydrogenase reacts reversibly with cyanide to produce a complex (E X NAD-CN) whose noncovalent interactions are similar to those in the E X NADH complex (where E is one-fourth of the tetrameric dehydrogenase). The reaction apparently is a simple bimolecular nucleophilic addition at the 4 position of the bound nicotinamide ring; viz., cyanide does not bind to the enzyme prior to reaction. The value of the dissociation constant for E X NAD-CN is about 1 X 10(-6) M and is independent of pH over the range of 6-8. The equilibrium constant for the reaction of cyanide with E X NAD is about 400-fold larger than that for the nonenzymic process after a statistical correction. This increment in Ke is accounted for by a 220-fold increase in the rate of the forward enzymic reaction (20 M-1 s-1) as compared with an approximately 2-fold decrease for the reverse process (9 X 10(-5) s-1). Thus, the increased value of the rate constant for bond formation in the enzymic reaction is attributed to an equilibrium binding effect that is translated almost entirely into a rate effect on that step (bond formation). Since the nonenzymic reaction is sensitive to solvent composition, this equilibrium binding effect likely is produced by environmental effects at the nicotinamide/dehydronicotinamide part of the coenzyme binding site on the enzyme.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

An affinity label for the regulatory dithiol of ribulose-5-phosphate kinase from maize (Zea mays).

Ribulose-5-phosphate kinase from maize (Zea mays) can exist in either a reduced, active form or an oxidized, inactive form. Reduced ribulose-5-phosphate kinase is rapidly and irreversibly inactivated by the dichlorotriazine dye Reactive Red 1 (Procion Red MX-2B), but the irreversible inactivation of the oxidized form of ribulose-5-phosphate kinase occurs at only 0.05% of this rate. The rate of inactivation of the reduced enzyme by Reactive Red 1 (apparent bimolecular rate constant 10(4)M-1 X s-1 at pH 7.4 and 25 degrees C) is several orders of magnitude greater than previous estimates of the rates of dye-mediated inactivation of other enzymes. The dye-dependent inactivation of the reduced enzyme is inhibited by Hg2+ or p-mercuribenzoate (thiol reagents that reversibly inhibit ribulose-5-phosphate kinase activity), or by ATP and ADP, the nucleotide substrates of the enzyme. Hydrolysed Reactive Red 1, which does not inactivate the enzyme, is a reversible inhibitor of ribulose-5-phosphate kinase. This inhibition is competitive with respect to ATP (Ki approximately 0.5 mM). The dye appears to act as an affinity label for the ATP/ADP-binding site by preferentially arylating a thiol residue generated during the reductive activation of the enzyme that is achieved by dithiothreitol or thioredoxin in vitro or during illumination of leaves.     

Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme.

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 X 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.     

Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump.

The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temperature-dependence measurements of the stationary pump current yielded an activation energy of approximately 100 kJ mol-1. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Na+ release. Simulating the data with a simple three-state model enabled us to estimate the turnover rate under saturating substrate concentrations, yielding rates (at pH 7.4) of approximately 60 s-1 and 200 s-1 at 24 degrees C and 36 degrees C, respectively.     

Purification and properties of malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum

Malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum is purified 50-fold to electrophoretic homogeneity. The purified enzyme crystallizes readily. Native malate dehydrogenase shows a relative molecular mass of 144 000. It is a tetramer of identical subunits with a relative molecular mass of 36 600. Malate dehydrogenase from Thermoplasma uses both NADH and NADPH as coenzyme to reduce oxaloacetate. The enzyme shows A-side (pro-R) stereospecificity for both coenzymes. The pH optimum for the reduction of oxaloacetate in the presence of NADH is found to be at pH 8.1. At pH 7.4 the Km value for oxaloacetate is found to be 5.6 microM while for NADH a value of 11.7 microM is found. The homogeneous enzyme shows a turnover number of kcat = 108 s-1.     

Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-galactosamine.

The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.     

Equilibrium and kinetic studies on the binding of gluconolactone to almond beta-glucosidase in the absence and presence of glucose

The binding of glucono-1,5-lactone (gluconolactone) with almond beta-glucosidase was studied at pH 5.0 and 25 degrees C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, delta Fmax, were determined to be 12.7 microM and 14.7%, respectively. From the study of the temperature dependence of Kd, delta G degrees, delta H degrees and delta S degrees for the binding were evaluated to be -6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25 degrees C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism: (formula; see text) which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k-1/k+1 = 4.5 mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2 = 94 s-1, k-2 = 0.36 s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.     

Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates.

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.     

Intermolecular charge transfer involving tryptophan, tyrosine and three electron-bonded intermediates derived from methionine

Oxidation processes of radiation-generated three-electron-bonded intermediates derived from methionine Met2[S+...S] and Met[S...X] (X=Cl,Br) were investigated through reaction with tryptophan and tyrosine, using the optical pulse radiolysis method. Bimolecular rate constants have been measured for the reactions Met2[S+...S] with TrpH(k=3.8 x 10(8) dm3 mol-1 s-1 and k = 4.9 X 10(8) dm3 mol-1 s-1 at at ph 1 and 4.3, respectively) and Met2[S+...S] with tyrosine, k=3.8 x 10(7) dm3 mol-1 s-1. Rate constants for intermolecular transformation of Met[S...Br] and Met[S...Cl] into TrpH+. or Trp. were also estimated. They varied from 4.7 X 10(8) dm3 mol-1 s-1 (bromide species) to 1.0 X 10(9)dm3 mol-1 s-1 (chloride species). It has also been established azide radicals N-6, N.3 in contrast to dihalide radicals (X-2) do not form transients of Met[S...X] (X = N3)-type. However, oxidation of N-3 by Met2[S+...S] occurs with a bimolecular rate constant of 2.8 X 10(8) dm3 mol-1 s-1. These results are discussed in the light of some equilibria which have been proposed earlier for methionine-halide systems.     

Evidence for catalytic dismutation of superoxide by cobalt(II) derivatives of bovine superoxide dismutase in aqueous solution as studied by pulse radiolysis.

By using the technique of pulse radiolysis to generate O2-., it is demonstrated that Co(II) derivatives of bovine superoxide dismutase in which the copper alone and both the copper and zinc of the enzyme have been substituted by Co(II), resulting in (Co,Zn)- and (Co,Co)-proteins, are capable of catalytically dismutating O2-. with 'turnover' rate constants of 4.8 X 10(6) dm3.s-1.mol-1 and 3.1 X 10(6) dm3.s-1.mol-1 respectively. The activities of the proteins are independent of the pH (7.4-9.4) and are about three orders of magnitude less than that of the native (Cu,Zn)-protein. The rate constants for the initial interaction of O2-. with the Co-proteins were determined to be (1.5-1.6) X 10(9) dm3.s-1.mol-1; however, in the presence of phosphate, partial inhibition is apparent [k approximately (1.9-2.3) X 10(8) dm3.s-1.mol-1]. To account for the experimental observations, two reaction schemes are presented, involving initially either complex-formation or redox reactions between O2-. and Co(II). This is the first demonstration that substitution of a metal into the vacant copper site of (Cu,Zn)-protein results in proteins that retain superoxide dismutase activity.     

Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator.

NADH chemistry ancillary to the oscillatory peroxidase-oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 +/- 0.2) x 105 M-1.s-1 and CpII to HRP at 1 x 105 M-1.s-1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 x 106 M-1.s-1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of alpha-NADH, not only the beta anomer as previously assumed. Rate constants of alpha- and beta-NADH reactions with CpI are (7.4 +/- 0.4) x 105 M-1.s-1, and (1.7 +/- 0.2) x 105 M-1.s-1, and with CpII are estimated as 5 x 104 M-1.s-1, and 4 x 104 M-1.s-1. Apparent rate constants of reduction of methylene blue (MB) to leuco-methylene blue (MBH) are 3.8 x 104 M-1.s-1 for NADH and 6.4 x 104 M-1.s-1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 +/- 0.2) x 103 M-1.s-1 All the rates were measured in 0.1 M acetate buffer, pH 5.1.     

Reaction of the 2',3'-dialdehyde derivative of NADPH at a nucleotide site of bovine liver glutamate dehydrogenase

The 2',3'-dialdehyde derivative of NADPH (oNADPH) acts as a coenzyme for the reaction catalyzed by bovine liver glutamate dehydrogenase. Incubation of 250 microM oNADPH with enzyme for 300 min at 30 degrees C and pH 8.0 yields covalent incorporation of 1.0 mol of oNADPH/mol of enzyme subunit. The modified enzyme has a functional catalytic site and is activated by ADP, but is no longer inhibited by high NADH concentrations and exhibits decreased sensitivity to GTP inhibition. Using the change in inhibition by 600 microM NADH or 1 microM GTP to monitor the reaction leads to rate constants of 44.0 and 41.5 min-1 M-1, respectively, suggesting that loss of inhibition by the two regulatory compounds results from reaction by oNADPH at a single location. The oNADPH incorporation is proportional to the decreased inhibition by 600 microM NADH or 1 microM GTP, extrapolating to less than 1 mol of oNADPH/mol of subunit when the maximum change in NADH or GTP inhibition has occurred. Modified enzyme is still 93% inhibited at saturating levels of GTP, although its K1 is increased 20-fold to 4.6 microM. The kinetic effects caused by oNADPH are not prevented by alpha-ketoglutarate, ADP, 5 mM NADH, or 200 microM GTP alone, but are prevented by 5 mM NADH with 200 microM GTP. Incorporation of oNADPH into enzyme at 255 min is 0.94 mol/mol of peptide chain in the absence of ligands but only 0.53 mol/mol of peptide chain in the presence of the protectants 5 mM NADH plus 200 microM GTP. These results indicate that oNADPH modifies specifically about 0.4-0.5 sites/enzyme subunit or about 3 sites/enzyme hexamer and that reaction occurs at a GTP-dependent inhibitory NADH site of glutamate dehydrogenase.     

Model studies for the direct effect of high-energy irradiation on DNA. Mechanism of strand break formation induced by laser photoionization of poly U in aqueous solution

Laser flash photolysis of polyuridylic acid (poly U) in anoxic aqueous solutions leads to biphotonic photoionization of the uracil moiety followed by the formation of single strand breaks (ssb). The rate constant for ssb formation (1.0 s-1, obtained from the slow component of conductivity increase at 23 degrees C and pH 6.8) increases with decreasing pH to 235 s-1 at pH 3.5. The activation energy (pre-exponential factor) was measured to be 66 kJ mol-1 (5 X 10(11) s-1) at pH 6.8. Addition of dithiothreitol (DTT) or glutathione (GSH) prevents ssb formation by reacting with a poly U intermediate (rate constant = 1.2 X 10(6) and 0.16 X 10(6) dm3 mol-1 s-1, respectively). Since with OH radicals as initiators very similar data have been obtained for the kinetics of ssb formation and for the reaction with DTT, we conclude that photoionization of the uracil moiety in poly U leads eventually to the same chemical pathway for ssb formation as that induced by OH radicals. Furthermore, we propose that protection by DTT and GSH occurs via H donation to the C-4' radicals of the sugar moiety of DNA and to the C-4' and the C-2' radicals of poly U.     

Inactivation-reactivation of two-electron reduced Escherichia coli glutathione reductase involving a dimer-monomer equilibrium

Glutathione reductase from Escherichia coli is inactivated when incubated with either NADPH or NADH. The process is inversely dependent on the enzyme concentration. Inactivation is rapid and monophasic with 1 microM NADPH and 1 nM enzyme FAD giving a t1/2 of 1 min. Complex formation between NADPH and the two-electron reduced enzyme (EH2) at higher levels of NADPH protects against rapid inactivation. NADP+, produced in a side reaction with oxygen, also protects by forming a complex with EH2. These complexes make analysis of the concentration dependence of the inactivation process difficult. Inactivation with NADH, where complexes do not interfere, is slower but can be analyzed more readily. With 152 microM NADH and 5.4 nM enzyme FAD, the time required for 50% inactivation is 17 min. The process is markedly biphasic, reaching the final inactivation level after 5-7 h. Analysis of the relationship between the final level of inactivation with NADH and the enzyme concentration indicates that inactivation is due to dissociation of the normally dimeric enzyme. Thus, the position of the dimer-monomer equilibrium between an active dimeric two-electron reduced species and an inactive monomeric two-electron reduced form determines the enzyme activity. An apparent equilibrium constant (Kd) for dissociation of dimer obtained from the anaerobic concentration dependent inactivation curves is 220 nM. Enzyme inactivated with NADH can be reactivated with glutathione, and the reactivation kinetics are second order, monomer-monomer over 75% of the reaction with an average apparent association rate constant (ka) of 13.1 (+/- 5.5) X 10(6) M-1 min-1.     

The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 A resolution by X-ray crystallography.

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones. DADH is the member of the short-chain dehydrogenases/reductases family (SDR) for which the largest amount of biochemical data has been gathered during the last three decades. The crystal structures of one binary form (NAD+) and three ternary complexes with NAD+.acetone, NAD+.3-pentanone and NAD+.cyclohexanone were solved at 2.4, 2.2, 1. 4 and 1.6 A resolution, respectively. From the molecular interactions observed, the reaction mechanism could be inferred. The structure of DADH undergoes a conformational change in order to bind the coenzyme. Furthermore, upon binding of the ketone, a region that was disordered in the apo form (186-191) gets stabilized and closes the active site cavity by creating either a small helix (NAD+. acetone, NAD+.3-pentanone) or an ordered loop (NAD+.cyclohexanone). The active site pocket comprises a hydrophobic bifurcated cavity which explains why the enzyme is more efficient in oxidizing secondary aliphatic alcohols (preferably R form) than primary ones. Difference Fourier maps showed that the ketone inhibitor molecule has undergone a covalent reaction with the coenzyme in all three ternary complexes. Due to the presence of the positively charged ring of the coenzyme (NAD+) and the residue Lys155, the amino acid Tyr151 is in its deprotonated (tyrosinate) state at physiological pH. Tyr151 can subtract a proton from the enolic form of the ketone and catalyze a nucleophilic attack of the Calphaatom to the C4 position of the coenzyme creating an NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accounts for the inactivation of the enzyme. The catalytic reaction proceeds in a similar way, involving the same amino acids as in the formation of the NAD-ketone adduct. The p Kavalue of 9-9.5 obtained by kinetic measurements on apo DADH can be assigned to a protonated Tyr151 which is converted to an unprotonated tyrosinate (p Ka7.6) by the influence of the positively charged nicotinamide ring in the binary enzyme-NAD+form. pH independence during the release of NADH from the binary complex enzyme-NADH can be explained by either a lack of electrostatic interaction between the coenzyme and Tyr151 or an apparent p Kavalue for this residue higher than 10.0.     

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.