308 research outputs found
Optimization of D-amino acid oxidase for low substrate concentrations--towards a cancer enzyme therapy.
No full textd-Amino acid oxidase (DAAO) has recently become of interest as a biocatalyst for industrial applications and for therapeutic treatments. It has been used in gene-directed enzyme prodrug therapies, in which its production of H(2)O(2) in tumor cells can be regulated by administration of substrate. This approach is limited by the locally low O(2) concentration and the high K(m) for this substrate. Using the directed evolution approach, one DAAO mutant was identified that has increased activity at low O(2) and d-Ala concentrations and a 10-fold lower K(m) for O(2). We report on the mechanism of this DAAO variant and on its cytotoxicity towards various mammalian cancer cell lines. The higher activity observed at low O(2) and d-Ala concentrations results from a combination of modifications of specific kinetic steps, each being of small magnitude. These results highlight the potential in vivo applicability of this evolved mutant DAAO for tumor therapy
Studies on Glycollate Oxidase from Pea Leaves : Determination of Stereospecificity and Mode of Inhibition by alpha-Hydroxybutynoate
No full textGlycollate oxidase (glycollate:oxygen oxidoreductase, EC 1.1.3.1) from Pisum sativum has an unusual absorption spectrum which suggests that the flavin N(3)-H function of the FMN coenzyme is ionized at pH 8.3. The enzyme is reduced rapidly by the substrate glycollate to yield a normal, reduced FMN coenzyme which is readily reoxidized by O2. No evidence for the occurrence of covalent intermediates during reduction, as observed upon reduction of L-Iactate oxidase from Mycobacterium smegmatis with the same substrate (Ghisla, S. and Massey, V. (1980) J. BioI. Chem. 255, 5688-5696), could be obtained. The enzyme was determined to be greater than 99.5% specific in the abstraction of the Re-hydrogen of glycollate. D-Lactate dehydrogenase from Lactobacillus Jeichmanii was shown to be only 97% selective for the Si-side in the reduction of glyoxylate. Glycollate oxidase was shown to be inhibited by α-hydroxybutynoate via covalent modification of the FMN coenzyme, in a fashion similar to that encountered with L-Iactate oxidase (Schonbrunn, A., Abeles, R.H., Walsh, Ch.T., Ghisla, S., Ogata, H. and Massey, V. (1976) Biochemistry 15, (1798-1807). This inhibitor serves both as substrate and inactivator of the enzyme.publishe
Studies on the catalytic mechanism of lactate oxidase : formation of enantiometric flavin-N(5)-glycollyl adducts via carbanion intermediates
No full textL-Lactate oxidase from Mycobacterium smegmatis reacts with the prochiral subsgtlryactoel late and forms a labile, catalytically competent glycollyl adduct in addition to a similar, but comparatively stable adduct (Massey, V., Ghisla, S., and Kieschke, K. (1980) J. Bid. Chem 255,2796-2806). The latter was isolated by Sephadex G-25 chromatography at 0-4°C and was also obtained from lactate oxidase, in which FMN had been replaced by the analogue 2-thio-FMN. The stable adduct is identical with the product obtained from illumination of the lactate oxidase●tartronate complex (Ghisla, S., Massey, V., and Choong, Y. S. (1979) J. Biol. Chem 254, 10662-10669) and thus has the structure of a glycollyl adduct to position N(5) of the reduced enzyme flavin. The stable adduct decays directly to oxidized enzyme and glycollate, with a t1/2 of 20 min at 25°C; the Arrhenius activation energy 21.4 kcal/mol. When the adduct is formed from reaction with [2,S- 2H] or with α-dideuteroglycollate, the decay reaction shows an isotope effect of 1.5. In contrast, no isotope effect is observed when the adduct is obtained from [2,R-2H]glycollate. Using [2,R-3H]- and [2,S-3H]glycollate, it is shown that the enzyme oxidizes catalytically the Re-hydrogen of this substrate, which is stereochemically equivalent to the α-hydrogen of L-lactate. On decay of the adduct, the Si-hydrogen bond of glycollate is (re)formed. This is demonstrated by the stereochemistry of glycollate obtained from decay of adduct formed photochemically from enzyme and [2-3H]- tartronate. The direct formation of a covalent glycollyl adduct at position N(5) of reduced FMN is interpreted as being equivalent to addition of a transient carbanion, which is formed by abstraction of a proton from the glycollate α position.publishe
Structural and kinetic analyses of the H121A mutant of cholesterol oxidase
No full textCholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a ≈100 mV decrease in the midpoint redox potential and a ≈40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024 18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 Å resolution (1 Å≡0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180° about χ1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.publishe
On the reaction of D-amino acid oxidase with dioxygen: O2 diffusion pathways and enhancement of reactivity.
No full textEvidence is accumulating that oxygen access in proteins is guided and controlled. We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoprotein D-amino acid oxidase (DAAO) that have a high affinity for dioxygen and are in close proximity to the flavin. With the goal of enhancing the reactivity of DAAO with oxygen, we have performed site-saturation mutagenesis at three positions that flank the putative oxygen channels and high-affinity sites. The most interesting variants at positions 50, 201 and 225 were identified by a screening procedure at low oxygen concentration. The biochemical properties of these variants have been studied and compared with those of wild-type DAAO, with emphasis on the reactivity of the reduced enzyme species with dioxygen. The substitutions at positions 50 and 225 do not enhance this reaction, but mainly affect the protein conformation and stability. However, the T201L variant shows an up to a threefold increase in the rate constant for reaction of O(2) with reduced flavin, together with a fivefold decrease in the K(m) for dioxygen. This effect was not observed when a valine is located at position 201, and is thus attributed to a specific alteration in the micro-environment of one high-affinity site for dioxygen (site B) close to the flavin that plays an important role in the storage of oxygen. The increase in O(2) reactivity observed for T201L DAAO is of great interest for designing new flavoenzymes for biotechnological applications
Revisitation of the betaCl-elimination reaction of D-amino acid oxidase: New interpretation of the reaction that sparked flavoprotein dehydrogenation mechanisms
No full textD-Amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl(-) from βCl-D-alanine via abstraction of the substrate α-H as H(+) (′′carbanion mechanism′′) (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J Biol Chem 246, 6855-6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme, but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (k(on) ≈ k(reverse)) between the complexes of oxidized enzyme-βCl-D-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O(2) the latter complex can partition between an oxidative half-reaction and elimination of Cl(-), which proceeds at a rate of ≈ 50 s(-1). This step forms a complex between oxidized enzyme and eneamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl(-) is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail
Properties of flavins where the 8-Methyl group is replaced by mercapto-Residues
No full textSulfur functions in position 8 of the flavin nucleus give rise to new modified flavin derivatives, which should prove useful as probes of the flavin binding domains of flavoproteins. Here, we report on some properties of 8-nor-8-alkylmercaptoflavins and 8-nor-8-mercaptoflavin which are readily formed by nucleophilic displacement by alkylmercaptides or sulfide, with 8-nor-8-chloroflavins as starting material. The new flavins are characterized by extensive shifts in spectral properties, with very high extinction coefficients. 8-nor-8-mercaptoriboflavin is easily and reversibly converted to its (-S-S-) dimer. Oxidation of the sulfur group by peracids forms first sulfoxides and then sulfones, in which the characteristic usual flavin spectrum is regained. A comparison of 8-SR-8-nor-flavins with 8-OR-8-nor-flavins (Ghisla, S., and Mayhew, S. G. (1976) Eur. J. Biochem 63, 373-390) indicates that in both classes of compounds, optical properties, ionization constants, and oxidation-reduction potentials follow similar patterns.publishe
Bioluminescence Emission from the Reaction of Luciferase-Flavin Mononucleotide Radical with Opt
No full textThe blue neutral luciferase flavin radical has been shown not to be in a catalytically significant equilibrium with species leading to emission of light [Kurfurst, M., Ghisla, S., Presswood, R., & Hastings, J. W. (1982) Eur. J. Biochem. 123, 355-361]. It is shown here that this radical can nevertheless react with 02¯● to form a species that is competent in light emission. From its properties, the species formed is deduced to be luciferase-FMNH 4a-hydroperoxide, a key intermediate in the normal luciferase reaction. Although it is concluded that this intermediate can undergo a reversible homolytic dissociation to yield free superoxide and the corresponding luciferase radical, the slowness of these steps precludes a catalytic significance for these pathways in the normal bioluminescent reaction.publishe
Reduced flavin : NMR investigation of N(5)-H exchange mechanism, estimation of ionisation constants and assessment of properties as biological catalyst
No full textBackground: The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. Results: N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. Conclusion: The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base
Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum
No full textThe kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s−1 and 105 s−1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s−1 for BCO and 232 s−1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Δ5-6→Δ4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s−1). With oxidized SCO and BCO the rate of isomerization is much faster (≈ 300 s−1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates ≈ 250 s−1 and 1.3 s−1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme product complexes.publishe
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