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1.
p-Hydroxybenzoate hydroxylase (PHBH) hydroxylates activated benzoates using NADPH as a reductant and O(2) as an oxygenating substrate. Because the flavin, when reduced, will quickly react with oxygen in either the presence or absence of a phenolic substrate, it is important to regulate flavin reduction to prevent the uncontrolled reaction of NADPH and oxygen to form H(2)O(2). Reduction is controlled by the protonation state of the aromatic substrate p-hydroxybenzoate (pOHB), which when ionized to the phenolate facilitates the movement of flavin between two conformations, termed "in" and "out". When the hydrogen bond network that provides communication between the substrate and solvent is disrupted by changing its terminal residue, His72, to Asn, protons from solution no longer equilibrate rapidly with pOHB bound to the active site [Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 1153-1158]. Thus, one population of the His72Asn enzyme reduces rapidly and has the phenolate form of pOHB bound at the active site and the flavin in the out conformation. The remaining population of the His72Asn enzyme reduces slowly and has the phenolic form of pOHB bound and the flavin in the in conformation. We have investigated the mechanisms of proton transfer between solvent and pOHB bound to the His72Asn form of the enzyme by double-mixing and single-mixing stopped-flow experiments. We find that, depending on the initial ionization state of bound pOHB and the new pH of the solution, the ionization/protonation of pOHB proceeds through the direct reaction of hydronium or hydroxide with the enzyme-ligand complex and leads to the conversion of one flavin conformation to the other. Our kinetic data indicate that the enzyme with the flavin in the in conformation reacts in two steps. Inspection of crystal structures suggests that the hydroxide ion would react at the re-face of the flavin, and its reaction with pOHB is limited by the movement of Pro293, a conserved residue in similar flavoprotein hydroxylases. We hypothesize that this type of breathing mode by the protein may have been used to compensate for the lack of an efficient proton-transfer network in ancestral hydroxylases, permitting useful catalysis prior to the emergence of specialized proton-transfer mechanisms. 相似文献
2.
Role of active site histidines in the two half-reactions of the aryl-alcohol oxidase catalytic cycle
A Hernández-Ortega F Lucas P Ferreira M Medina V Guallar AT Martínez 《Biochemistry》2012,51(33):6595-6608
The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k(cat)/K(m(Al)) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O(2) constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH(-) to O(2), without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H(+) proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H(2)O(2). No solvent KIE was found for O(2) reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k(cat)/K(m(Ox)), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations. 相似文献
3.
The FAD of p-hydroxybenzoate hydroxylase (PHBH) is known to exist in two conformations. The FAD must be in the in-position for hydroxylation of p-hydroxybenzoate (pOHB), whereas the out-position is essential for reduction of the flavin by NADPH. In these investigations, we have used 8-mercapto-FAD and 8-hydroxy-FAD to probe the movement of the flavin in catalysis. Under the conditions employed, 8-mercapto-FAD (pK(a) = 3.8) and 8-hydroxy-FAD (pK(a) = 4.8) are mainly anionic. The spectral characteristics of the anionic forms of these flavins are very sensitive to their environment, making them sensitive probes for detecting movement of the flavin during catalysis. With these flavin analogues, the enzyme hydroxylates pOHB efficiently, but at a rate much slower than that of enzyme with FAD. Reaction of oxygen with reduced forms of these modified enzymes in the absence of substrate appears to proceed through the formation of the flavin-C4a-hydroperoxide intermediate, as with normal enzyme, but the decay of this intermediate is so fast compared to its formation that very little accumulates during the reaction. However, after elimination of H2O2 from the flavin-C4a-hydroperoxide, a perturbed oxidized enzyme spectrum is observed (Eox*), and this converts slowly to the spectrum of the resting oxidized form of the enzyme (Eox). In the presence of pOHB, PHBH reconstituted with 8-mercapto-FAD also shows the additional oxidized intermediate (Eox*) after the usual oxygenated C4a-intermediates have formed and decayed in the course of the hydroxylation reaction. This Eox* to Eox step is postulated to be due to flavin movement. Furthermore, binding of pOHB to resting (Eox) follows a three-step equilibrium mechanism that is also consistent with flavin movement being the rate-limiting step. The rate for the slowest step during pOHB binding is similar to that observed for the conversion of Eox* to Eox during the oxygen reaction in the absence or presence of substrate. Steady-state kinetic analysis of PHBH substituted with 8-mercapto-FAD demonstrated that the apparent k(cat) is also similar to the rate of Eox* conversion to Eox. Presumably, the protein environment surrounding the flavin in Eox* differs slightly from that of the final resting form of the enzyme (Eox). 相似文献
4.
The intrinsic isotope effect on the reduction of the FAD-containing dehydrogenase electron transferase, adrenodoxin reductase, by (4S)-[2H]NADPH has been determined to be 7.1 to 7.7. The replacement of FAD by a series of FAD analogs at the active site of adrenodoxin reductase with oxidation-reduction potentials which vary over a range of 212 mV has made it possible to extrapolate to this limiting value from the variation in the observed isotope effect on Vmax with flavin midpoint potential. Stop-flow studies which allow the direct determination of the intrinsic isotope effect on the reductive half-reaction corroborate this result. During the steady state reduction of ferricyanide by the native enzyme under conditions of Vmax, this isotope effect is almost fully expressed (VH/VD = 6.7 to 6.8). In contrast, we observe a dramatic attenuation of the intrinsic isotope effect (due to hydride transfer to flavin) when the oxidative half-reaction is mediated by the natural acceptor protein, the 2Fe/2S ferredoxin, adrenodoxin. In a coupled three-protein system, the adrenodoxin-mediated reductions of both the artificial electron acceptor, cytochrome c, and the physiological electron acceptor, cytochrome P-450scc, by adrenodoxin reductase occur at similar rates and with similar kinetic isotope effects (1.9 to 2.0) when (4S)-[2H]NADPH is the reductant. We infer similar mechanisms for the reduction of both cytochromes. These results are in agreement with previous studies (Lambeth, J.D., and Kamin, H. (1979) J. Biol. Chem. 254, 2766-2774) which show that the reductive half-reaction is not solely rate-determining in adrenodoxin-mediated processes. The observation of a linear free energy relationship between Vmax and the flavin midpoint potential during steady state reduction of ferricyanide confirms that the reductive half-reaction is rate-determining in this assay. The relationship between Vmax and flavin midpoint potential in reactions which require adrenodoxin suggests that the midpoint potential of native adrenodoxin reductase has been optimized. Thus, the apoenzyme of adrenodoxin reductase tailors the midpoint potential of bound FAD in order to balance the activation energies of the reductive and oxidative half-reactions. 相似文献
5.
Apo-p-hydroxybenzoate hydroxylase was reconstituted using 2'-fluoro-2'-deoxy-arabino-FAD, a synthetic flavin in which the hydroxyl of the 2'-center of the ribityl chain was replaced with fluorine in an inverted configuration. The absorbance spectral changes caused by the binding of either p-hydroxybenzoate (pOHB) or 2,4-dihydroxybenzoate (2,4-diOHB) indicated that the isoalloxazine of the artificial flavin adopts the more solvent-exposed "out" conformation rather than the partially buried "in" conformation near the aromatic substrate. In contrast, the flavin of the natural enzyme adopts the in conformation when pOHB is bound. Much of the behavior of the artificial enzyme can be rationalized in light of the preference of the flavin for the out conformation, including the weaker binding of pOHB, the tighter binding of 2,4-diOHB, and the slower reactions involved in the hydroxylation of pOHB and 2,4-diOHB. Particularly noteworthy is the enhancement of the reduction of the flavin by NADPH when pOHB is bound to the active site, consistent with the recent finding that the reaction occurs when the flavin adopts the out conformation (Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 1153-1158). Thus, whereas the change that induces the out conformation is detrimental to the oxidative half-reaction, it improves the reductive half-reaction, showing that the control of the flavin position in p-hydroxybenzoate hydroxylase represents a compromise between the conflicting needs of two chemically disparate half-reactions, and demonstrating that the 2'-hydroxyl of FAD can serve as a critical control element in flavoenzyme catalysis. 相似文献
6.
The p67(phox) activation domain regulates electron flow from NADPH to flavin in flavocytochrome b(558). 总被引:1,自引:0,他引:1
Y Nisimoto S Motalebi C H Han J D Lambeth 《The Journal of biological chemistry》1999,274(33):22999-23005
An activation domain in p67(phox) (residues within 199-210) is essential for cytochrome b(558)-dependent activation of NADPH superoxide (O2(-.)) generation in a cell-free system (Han, C.-H., Freeman, J. L. R., Lee, T., Motalebi, S. A., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 16663-16668). To determine the steady state reduction flavin in the presence of highly absorbing hemes, 8-nor-8-S-thioacetamido-FAD ("thioacetamido-FAD") was reconstituted into the flavocytochrome, and the fluorescence of its oxidized form was monitored. Thioacetamido-FAD-reconstituted cytochrome showed lower activity (7% versus 100%) and increased steady state flavin reduction (28 versus <5%) compared with the enzyme reconstituted with native FAD. Omission of p67(phox) decreased the percent steady state reduction of the flavin to 4%, but omission of p47(phox) had little effect. The activation domain on p67(phox) was critical for regulating flavin reduction, since mutations in this region that decreased O2(-.) generation also decreased the steady state reduction of flavin. Thus, the activation domain on p67(phox) regulates the reductive half-reaction for FAD. This reaction is comprised of the binding of NADPH followed by hydride transfer to the flavin. Kinetic deuterium isotope effects along with K(m) values permitted calculation of the K(d) for NADPH. (R)-NADPD but not (S)-NADPD showed kinetic deuterium isotope effects on V and V/K of about 1.9 and 1.5, respectively, demonstrating stereospecificity for the R hydride transfer. The calculated K(d) for NADPH was 40 microM in the presence of wild type p67(phox) and was approximately 55 microM using the weakly activating p67(phox)(V205A). Thus, the activation domain of p67(phox) regulates the reduction of FAD but has only a small effect on NADPH binding, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD. 相似文献
7.
The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide, des-myo-inositol mycothione disulfide, has been determined. An analysis of the pH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K(DIMSSM), yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K(NADPH). Proton inventory studies (V versus mole fraction of D(2)O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady-state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction. 相似文献
8.
Enterococcus faecalis dihydroorotate dehydrogenase B is a heterodimer of 28 and 33 kDa encoded by the pyrK and pyrDb genes. Both subunits copurify during all chromatographic steps, and, as determined by HPLC, one FMN and one FAD are bound per heterodimer. The enzyme catalyzes efficient oxidation of 4-S-NADH by orotate. Isotope effect and pH data suggest that reduction of flavin by NADH at the PyrK site is only partially rate limiting with no kinetically significant proton transfer occurring in the reductive half-reaction; therefore, a group exhibiting a pK of 5.7 +/- 0.2 represents a residue involved in binding of NADH rather than in catalysis. The reducing equivalents are shuttled between the NADH-oxidizing flavin in PyrK and the orotate-reacting flavin in PyrDb, by iron-sulfur centers through flavin semiquinones as intermediates. A solvent kinetic isotope effect of 2.5 +/- 0.2 on V is indicative of rate-limiting protonation in the oxidative half-reaction and most likely reflects the interaction between the isoalloxazine N1 of the orotate-reducing flavin and Lys 168 (by analogy with L. lactis DHODase A). The oxidative half-reaction is facilitated by deprotonation of the group(s) with pK(s) of 5.8-6.3 and reflects either deprotonation of the reduced flavin or binding of orotate; this step is followed by hydride transfer to C6 and general acid-assisted protonation (pK of 9.1 +/- 0.2) at C5 of the product. 相似文献
9.
EBP1-catalyzed reduction of alpha,beta-unsaturated ketones and aldehydes is proposed to proceed via transfer of hydride from the flavin to the beta-position of the olefinic bond, concomitant with or followed by uptake of a proton at the alpha-position. Structural analysis suggests that this proton is donated from Tyr206, and, hence, a protein was constructed in which it was replaced by phenylalanine. The mutation results in a slightly less stable protein than the wild type that nevertheless retains the fundamental flavin and phenol binding properties of EBP1 characterized previously. The pH profile for binding of phenol was characterized over the pH range 6.5-9.5 and was found to be simpler than that for the wild-type enzyme. Most importantly, a pK(a) of 8.7 that is perturbed to 9.4 upon binding of phenol to the wild-type enzyme is missing in the mutant, allowing assignment of this pK(a) to the Y206 hydroxyl group. Additionally, the pK(a) of phenol is further lowered from its value of 10.0 in solution to approximately 6.4 in the active site of the mutant, as compared to 7.1 in the wild type. Together, these perturbations lead to an increase of approximately 35-fold in the binding affinity of the mutant for phenol at high pH relative to the affinity of the wild-type enzyme. As expected, the mutation has little effect on the reductive half-reaction, in which a hydride equivalent is transferred from NADPH to the flavin. In contrast, the reduction of trans-2-hexenal by the reduced enzyme is significantly affected. The results indicate formation of a previously unobserved charge-transfer (CT) complex following formation of the Michaelis complex between substrate and reduced enzyme and preceding reduction of the substrate, which occurs at a greatly reduced rate (>/=440-fold) relative to wild type. Thus, while the oxidative half-reaction with wild-type enzyme is limited by the rate of formation of the CT complex, it is the chemical step that is rate-limiting in the reaction with EBP1:Y206F, consistent with the role of this residue as a general acid. 相似文献
10.
The neuronal nitric oxide synthase (nNOS) basal and calmodulin- (CaM-) stimulated reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) follow ping-pong mechanisms [Wolthers and Schimerlik (2001) Biochemistry 40, 4722-4737]. Primary deuterium [NADPH(D)] and solvent deuterium isotope effects on the kinetic parameters were studied to determine rate-limiting step(s) in the kinetic mechanisms for the two substrates. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Values for (D)V and (D)(V/K)(NADPH) were similar for the basal (1.3-1.7) and CaM-stimulated (1.5-2.1) reduction of DCIP, while (D)V (2.1-2.8) was higher than (D)(V/K)(NADPH) (1.1-1.5) for cytochrome c(3+) reduction with and without CaM. This suggests that the rate of the reductive half-reaction (NADPH oxidation) rather than that of the oxidative half-reaction (reduction of DCIP or cytochrome c(3+)) limits the overall reaction rate. A value for (D)(V/K)(NADPH) close to 1 indicates the intrinsic isotope effect on hydride transfer is suppressed by a slower step in the reductive half-reaction. The oxidative half-reaction is insensitive to NADPD isotope effects as both (D)(V/K)(DCIP) and (D)(V/K)(cytc) equal 1 within experimental error. Large solvent kinetic isotope effects (SKIE) observed for (V/K)(cytc) for basal (approximately 8) and CaM-stimulated (approximately 31) reduction of cytochrome c(3+) suggest that proton uptake from the solvent limits the rate of the oxidative half-reaction. This step does not severely limit the overall reaction rate as (D2O)V equaled 2 and (D2O)(V/K)(NADPH) was between 0.9 and 1.3 for basal and CaM-stimulated cytochrome c(3+) reduction. 相似文献
11.
Human methionine synthase reductase (MSR) is a key enzyme in folate and methionine metabolism as it reactivates the catalytically inert cob(II)alamin form of methionine synthase (MS). Electron transfer from MSR to the cob(II)alamin cofactor coupled with methyl transfer from S-adenosyl methionine returns MS to the active methylcob(III)alamin state. MSR contains stoichiometric amounts of FAD and FMN, which shuttle NADPH-derived electrons to the MS cob(II)alamin cofactor. Herein, we have investigated the pre-steady state kinetic behavior of the reductive half-reaction of MSR by anaerobic stopped-flow absorbance and fluorescence spectroscopy. Photodiode array and single-wavelength spectroscopy performed on both full-length MSR and the isolated FAD domain enabled assignment of observed kinetic phases to mechanistic steps in reduction of the flavins. Under single turnover conditions, reduction of the isolated FAD domain by NADPH occurs in two kinetically resolved steps: a rapid (120 s(-1)) phase, characterized by the formation of a charge-transfer complex between oxidized FAD and NADPH, is followed by a slower (20 s(-1)) phase involving flavin reduction. These two kinetic phases are also observed for reduction of full-length MSR by NADPH, and are followed by two slower and additional kinetic phases (0.2 and 0.016 s(-1)) involving electron transfer between FAD and FMN (thus yielding the disemiquinoid form of MSR) and further reduction of MSR by a second molecule of NADPH. The observed rate constants associated with flavin reduction are dependent hyperbolically on NADPH and [4(R)-2H]NADPH concentration, and the observed primary kinetic isotope effect on this step is 2.2 and 1.7 for the isolated FAD domain and full-length MSR, respectively. Both full-length MSR and the separated FAD domain that have been reduced with dithionite catalyze the reduction of NADP+. The observed rate constant of reverse hydride transfer increases hyperbolically with NADP+ concentration with the FAD domain. The stopped-flow kinetic data, in conjunction with the reported redox potentials of the flavin cofactors for MSR [Wolthers, K. R., Basran, J., Munro, A. W., and Scrutton, N. S. (2003) Biochemistry, 42, 3911-3920], are used to define the mechanism of electron transfer for the reductive half-reaction of MSR. Comparisons are made with similar stopped-flow kinetic studies of the structurally related enzymes cytochrome P450 reductase and nitric oxide synthase. 相似文献
12.
Basran J Harris RJ Sutcliffe MJ Scrutton NS 《The Journal of biological chemistry》2003,278(45):43973-43982
The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperature-dependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the alpha/beta unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperature-dependent reaction rates, again consistent with ground-state tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme. 相似文献
13.
Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine with the formation of an aldehyde intermediate. In the first oxidation reaction, the alcohol substrate is initially activated to its alkoxide via proton abstraction. The substrate is oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. In this study, we have mutagenized an active site serine proximal to the C(4a) and N(5) atoms of the flavin and investigated the reactions of proton and hydride transfers by using substrate and solvent kinetic isotope effects. Replacement of Ser101 with threonine, alanine, cysteine, or valine resulted in biphasic traces in anaerobic reductions of the flavin with choline investigated in a stopped-flow spectrophotometer. Kinetic isotope effects established that the kinetic phases correspond to the proton and hydride transfer reactions catalyzed by the enzyme. Upon removal of Ser101, there is an at least 15-fold decrease in the rate constants for proton abstraction, irrespective of whether threonine, alanine, valine, or cysteine is present in the mutant enzyme. A logarithmic decrease spanning 4 orders of magnitude is seen in the rate constants for hydride transfer with increasing hydrophobicity of the side chain at position 101. This study shows that the hydrophilic character of a serine residue proximal to the C(4a) and N(5) flavin atoms is important for efficient hydride transfer. 相似文献
14.
Vibrio harveyi NADPH:FMN oxidoreductase P (FRP(Vh)) is a homodimeric enzyme having a bound FMN per enzyme monomer. The bound FMN functions as a cofactor of FRP(Vh) in transferring reducing equivalents from NADPH to a flavin substrate in the absence of V. harveyi luciferase but as a substrate for FRP(Vh) in the luciferase-coupled bioluminescent reaction. As part of an integral plan to elucidate the regulation of functional coupling between FRP(Vh) and luciferase, this study was carried out to characterize the equilibrium bindings, reductive potential, and the reversibility of the reduction of the bound FMN in the reductive half-reaction of FRP(Vh). Results indicate that, in addition to NADPH binding, NADP(+) also bound to FRP(Vh) in either the oxidized (K(d) 180 microM) or reduced (K(d) 230 microM) form. By titrations with NADP(+) and NADPH and by an isotope exchange experiment, the reduction of the bound FMN by NADPH was found to be readily reversible (K(eq) = 0.8). Hence, the reduction of FRP(Vh)-bound FMN is not the committed step in coupling the NADPH oxidation to bioluminescence. To our knowledge, such an aspect of flavin reductase catalysis has only been clearly established for FRP(Vh). Although the reductive potentials and some other properties of a R203A variant of FRP(Vh) and an NADH/NADPH-utilizing flavin reductase from Vibrio fischeri are quite similar to that of the wild-type FRP(Vh), the reversal of the reduction of bound FMN was not detected for either of these two enzymes. 相似文献
15.
Previous work from this laboratory has established that the NADPH-menadione oxidoreductase reaction catalyzed by methylenetetrahydrofolate reductase from pig liver proceeds by Ping Pong Bi Bi kinetics and that the reductive half-reaction is rate limiting in steady-state turnover. We have now shown that methylenetetrahydrofolate reductase stereo-specifically removes the pro-S hydrogen from the 4-position of NADPH. During the oxidation of [4(S)-3H]NADPH, we observed a kinetic isotope on V/KNADPH of 10.8 +/- 0.4. When comparing the rates of oxidation of [4(S)-2H]NADPH and [4(S)-1H]NADPH, we measure kinetic isotope effects on V of 4.78 +/- 0.15 and on V/KNADPH of 4.54 +/- 0.59. When oxidation of [4(R)-2H]NADPH and [4(R)-1H]NADPH is compared, the secondary kinetic isotope effect on V is 1.04 +/- 0.01. When the NADPH-menadione oxidoreductase reaction is catalyzed in tritiated water, no incorporation of solvent tritium into residual NADPH is observed. We conclude from these observations that the oxidation of NADPH is largely or entirely rate limiting in the reductive half-reaction and, hence, in NADPH-menadione oxidoreductase turnover at saturating menadione concentration. In the presence of saturating NADPH, the flavin reduction proceeds with a rate constant of 160 S-1, which is at least 29-fold slower than estimates of the lower limit for the diffusion-limited rate constant characterizing NADPH binding to the enzyme under physiological conditions. Albery & Knowles have defined criteria for perfection in enzyme catalysis [Albery, W. J., & Knowles, J.R. (1976) Biochemistry 15, 5631-5640].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
17.
Leskovac V Trivić S Wohlfahrt G Kandrac J Pericin D 《The international journal of biochemistry & cell biology》2005,37(4):731-750
Glucose oxidase from the mold Aspergillus niger (EC 1.1.3.4) oxidizes beta-D-glucose with a wide variety of oxidizing substrates. The substrates were divided into three main groups: molecular oxygen, quinones, and one-electron acceptors. The kinetic and chemical mechanism of action for each group of substrates was examined in turn with a wide variety of kinetic methods and by means of molecular modeling of enzyme-substrate complexes. There are two proposed mechanisms for the reductive half-reaction: hydride abstraction and nucleophilic attack followed by deprotonation. The former mechanism appears plausible; here, beta-D-glucose is oxidized to glucono-delta-lactone by a concerted transfer of a proton from its C1-hydroxyl to a basic group on the enzyme (His516) and a direct hydride transfer from its C1 position to the N5 position in FAD. The oxidative half-reaction proceeds via one- or two-electron transfer mechanisms, depending on the type of the oxidizing substrate. The active site of the enzyme contains, in addition to FAD, three amino acid side chains that are intimately involved in catalysis: His516 with a pK(a)=6.9, and Glu412 with pK(a)=3.4 which is hydrogen bonded to His559, with pK(a)>8. The protonation of each of these residues has a strong influence on all rate constants in the catalytic mechanism. 相似文献
18.
The transient kinetics of the reaction of the estrogen binding protein (EBP1) from Candida albicans in which hydride is transferred from NADPH to trans-2-hexenal (HXL) in two half-reactions were analyzed using UV-visible spectrophotometric and fluorometric stopped-flow techniques. The simplest model of the first half-reaction involves four steps including very rapid, tight binding (K(d) = 50 nM) characterized by loss of NADPH fluorescence, subsequent rapid formation of a charge-transfer complex between NADPH and oxidized enzyme-bound flavin mononucleotide (FMN) cofactor, followed by rate-limiting reduction of the FMN, and finally dissociation of the oxidized pyridine nucleotide. The UV-visible absorbance behavior of this half-reaction is described by two apparent phases, with k(f)(obs) = 355 +/- 6 and k(s)(obs) = 3.30 +/- 0.03 s(-)(1), for the fast and slow phases, respectively. The reaction has also been evaluated in terms of the full, multi-equilibrium reaction scheme, and microscopic rate constants that lead to the observed behavior have been determined through convergent experimental techniques and computer simulation. Significant intrinsic kinetic isotope effects were noted on both the bond cleavage step and the preceding formation of the charge-transfer complex. The enzyme is reoxidized by transfer of hydride from the FMN to HXL in the second half-reaction that appears to consist of substrate binding to form a Michaelis-type complex and the subsequent chemical step. Characterization of the reaction in this simple manner allows determination of an apparent K(d) = 100 +/- 9 microM for the reduced enzyme.HXL complex. 相似文献
19.
Khan H Harris RJ Barna T Craig DH Bruce NC Munro AW Moody PC Scrutton NS 《The Journal of biological chemistry》2002,277(24):21906-21912
The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP. 相似文献
20.
p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al. (2005) Arch. Biochem. Biophys. 433, 297-311]. The open conformation is important for substrate binding and product release, the in conformation for reaction with oxygen and hydroxylation, and the out conformation for the reduction of FAD by NADPH. The open conformation is similar to the structure of Arg220Gln-PHBH in which the backbone peptide loop of residues 43-46, located on the si side of the flavin, is rotated. In this paper, we examine the structure and properties of the Ala45Gly-PHBH mutant enzyme. The crystal structure of the Ala45Gly enzyme is an asymmetric dimer, with one monomer similar (but not identical) to wild-type PHBH, while the other monomer has His72 flipped into solvent and replaced with Glu73 as one of several changes in the structure. The two structures correlate with evidence from kinetic studies for two forms of Ala45Gly-PHBH. One form of the enzyme dominates turnover and hydroxylates, while the other contributes little to turnover and fails to hydroxylate. Ala45Gly-PHBH favors the in conformation over alternative conformations. The effect of this mutation on the structure and function of PHBH illustrates the importance of the si side loop in the conformational state of PHBH and, consequently, the function of the enzyme. This work demonstrates some general principles of how enzymes use conformational movements to allow both access and egress of substrates and product, while restricting access to the solvent at a critical stage in catalysis. 相似文献