Substrate specificity of SIRT1-catalyzed lysine N-deacetylation reaction probed with the side chain modified N-acetyl-lysine analogs

Peptides containing l-N^ε-acetyl-lysine (l-AcK) or its side chain modified analogs were prepared and assayed using SIRT1, the prototypical human silent information regulator 2 (Sir2) enzyme. While previous studies showed that the side chain acetyl group of l-AcK can be extended to bulkier acyl groups for Sir2 (including SIRT1)-catalyzed lysine N^ε-deacylation reaction, our current study suggested that SIRT1-catalyzed deacetylation reaction had a very stringent requirement for the distance between the α-carbon and the side chain acetamido group, with that found in l-AcK being optimal. Moreover, our current study showed that SIRT1 catalyzed the stereospecific deacetylation of l-AcK versus its d-isomer. The results from our current study shall constitute another piece of important information to be considered when designing inhibitors for SIRT1 and Sir2 enzymes in general.     

Kinetics and activation thermodynamics of methane monooxygenase compound Q formation and reaction with substrates

The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of methane by Q exceeds that of many other hydrocarbons with weaker C-H bonds.     

Mechanism-based inactivation of the flavoprotein cyclohexanone monooxygenase by S oxygenation

The FAD-dependent enzyme cyclohexanone oxygenase carries out biological Baeyer-Villiger oxidations of ketones and aldehydes with O₂ as cosubstrate. Sulfides are enzymically oxygenated to sulfoxides but thiolactones appear to be enzymically processed to acyl sulfoxides which are reactive acylating and then sulfenylating agents, inducing mechanism-based inactivation.     

Methane monooxygenase: purification and properties of flavoprotein component

An anaerobic procedure was developed for the purification of the flavin:NADH oxidoreductase (flavoprotein) component of methane monooxygenase to homogeneity. The molecular weight of the flavoprotein determined by gel filtration was about 40,000, and by sedimentation equilibrium analysis, about 38,000. The purified flavoprotein is a monomeric protein with a sedimentation constant (S20,W) value of about 2.1 S. The absorption spectrum of the flavoprotein has a peak at 460 nm and shoulder at 395 nm. The fluorescent excitation and emission spectra of the fluorescent component of flavoprotein had peaks at 450, 370, and 530 nm, respectively. A FAD was identified as a prosthetic group of flavoprotein by thin-layer chromatography. The flavoprotein contained about 1 mol of FAD and 2 mol each of iron and acid-labile sulfide per mole of protein. The flavoprotein was directly reduced by NADH under anaerobic conditions. The formation of neutral flavin semiquinone was detected during anaerobic titration of flavoprotein by NADH and also as a free radical signal at a g value of 2.004 by EPR spectroscopy. The iron sulfur cluster has g values of 2.04, 1.96, and 1.87, yielding a g average of 1.96, characteristic of a Fe2S2 center. Antibody prepared against the flavoprotein reacted with flavoprotein and inhibited methane monooxygenase activity.     

Studies on the specificity toward aldehyde substrates and steady-state kinetics of xanthine oxidase

The aldehyde specificity of xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been reinvestigated. The biogenic aldehydes and succinate semialdehyde are reasonable substrates for xanthine oxidase. Pyrophosphate, which binds to xanthine oxidase, does not seem to affect significantly the enzyme's catalytic activity. The steady-state parameters for the oxidation of several substrates by xanthine oxidase and oxygen have been determined. Formaldehyde differs from xanthine and other aldehydes in phi 2, the parameter describing the reaction with oxygen. Substrate inhibition has been studied at high concentrations of xanthine with oxygen as the electron acceptor. The inhibition is hyperbolic and uncompetitive with respect to oxygen. This is possibly due to rate-limiting product release from molybdenum(IV) being slower than from molybdenum(VI).     

Mechanisms of reaction of some flavoprotein enzymes with oxygen

Biochemical investigations of the properties of free flavins and of flavoproteins have shown that reduction usually occurs in two stages, with the intermediate formation of semiquinones in the case of free flavins. Flavoproteins often show spectroscopically similar intermediates, when partially reduced with substrate. These may, however, be enzyme-product complexes. Detailed investigation of individual flavoprotein enzymes has shown examples in which catalysis involves transition of the enzyme between oxidized and fully reduced forms (glucose oxidase), between oxidized and intermediate forms (D-amino acid oxidase), and intermediate and fully reduced forms (TPNH—cytochrome c reductase). Further, examples are known in which both intermediate and reduced forms react with oxygen, in which only one reacts, while in TPNH—cytochrome c reductase neither the intermediate nor the reduced form reacts with molecular oxygen. The physiological significance of these complex findings is uncertain, partly because it is not known whether purified flavoproteins occur in the same form in the tissues. It seems unlikely, however, that flavoproteins make a major contribution to the respiratory exchange of mammals.     

The reaction of p-nitrophenyl acetate with lysine and lysine derivatives

The kinetics of the reaction of p-nitrophenyl acetate with lysine and its derivatives has been investigated in water-dioxane solutions over the pH range 8.1–10.1. The reaction was found to be second order, and the unprotonated amino group was shown to be the reactive species. Intrinsic second-order rate constants were calculated.     

Studies of the oxidative half-reaction of anthranilate hydroxylase (deaminating) with native and modified substrates

The oxidative half-reactions of anthranilate hydroxylase (EC 1.14.12.2) were examined in the presence of anthranilate and modified substrates. C(4a)-Hydroperoxyflavin (C(4a)-FlOOH) and C(4a)-hydroxyflavin (C(4a)-FlOH) intermediates were detected in oxidative reactions with all substrates. Thus, the oxygenation reactions of the enzyme are similar to those of flavoprotein hydroxylases that convert phenolic compounds to catechols. These observations support a mechanism proposed for this enzyme (Powlowski, J. B., Dagley, S., Massey, V., and Ballou, D. P. (1987) J. Biol. Chem. 262, 69-74) involving nucleophilic attack of the substrate on C(4a)-FlOOH, and formation of an imine intermediate that is subsequently hydrolyzed. Anthranilate hydroxylase is therefore a typical flavoprotein hydroxylase with the added capacity of hydrolyzing imine intermediates. Fluorine substituents on the aromatic ring decreased the rate of conversion of C(4a)-FlOOH to C(4a)-FlOH, as predicted by this mechanism. Hydroxylation of 3-fluoro- and 3-methylanthranilates resulted in the formation of nonaromatic products that appeared to stabilize the C(4a)-FlOH. No evidence was found for a high extinction intermediate (intermediate II) (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563) under conditions where it was readily detected with other flavoprotein hydroxylases. It was shown that the spectra of the nonaromatic products (which are quinonoid forms) could not be summed with the spectra of C(4a)-hydroxyflavin to obtain that of a putative intermediate II, thus ruling out that explanation for previous observations of II.     

Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase

Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.     

Two structures of an N-hydroxylating flavoprotein monooxygenase: ornithine hydroxylase from Pseudomonas aeruginosa

The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the side chain amine of ornithine, which is subsequently formylated to generate the iron-chelating hydroxamates of the siderophore pyoverdin. PvdA belongs to the class B flavoprotein monooxygenases, which catalyze the oxidation of substrates using NADPH as the electron donor and molecular oxygen. Class B enzymes include the well studied flavin-containing monooxygenases and Baeyer-Villiger monooxygenases. The first two structures of a class B N-hydroxylating monooxygenase were determined with FAD in oxidized (1.9 Å resolution) and reduced (3.03 Å resolution) states. PvdA has the two expected Rossmann-like dinucleotide-binding domains for FAD and NADPH and also a substrate-binding domain, with the active site at the interface between the three domains. The structures have NADP(H) and (hydroxy)ornithine bound in a solvent-exposed active site, providing structural evidence for substrate and co-substrate specificity and the inability of PvdA to bind FAD tightly. Structural and biochemical evidence indicates that NADP⁺ remains bound throughout the oxidative half-reaction, which is proposed to shelter the flavin intermediates from solvent and thereby prevent uncoupling of NADPH oxidation from hydroxylated product formation.     

The specificity and multiplicity of human placental xenobiotic-metabolizing monooxygenase system studied by potential substrates, inhibitors and gel electrophoresis

The specificity of the placental monooxygenase system to metabolize foreign compounds was studied by using different potential substrates and inhibitors and by performing electrophoresis of placental microsomes. Placental preparations from smokers catalyzed benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation and 2,5-diphenyloxazole hydroxylation, but not biphenyl hydroxylation at 2-, 3- or 4-carbon, aldrin epoxidation to dieldrin or coumarin hydroxylation or aminopyrine N-demethylation. Enzyme activities were inhibited by alpha-naphthoflavone, but to a much lesser extent by SKF 525-A or metyrapone. Correlations between the metabolism of benzo(a)pyrene, 7-ethoxycoumarin and 2,5-diphenyloxazole were highly significant. There was a clear difference in Michaelis-Menten constant of 7-ethoxycoumarin O-deethylation between placentas from smokers and nonsmokers. Gel electrophoresis revealed that protein bands of placental microsomes in the region of cytochrome P-450 enzymes were less prominent than those of rat liver microsomes, a finding that accorded with the relative amounts of cytochrome P-450. There were no consistent differences in the electrophoretic pattern between placentas of variable benzo(a)pyrene hydroxylase activities. Results show that the human placental monooxygenase system is restricted in substrate specificity, that there may be a qualitative difference between smokers and nonsmokers and that the increase in several enzyme activities by cigarette smoking cannot be detected by the standard gel electrophoresis.     

Tigecycline is modified by the flavin-dependent monooxygenase TetX

The clinical use of tetracycline antibiotics has decreased due to the emergence of efflux and ribosomal protection-based resistance mechanisms. Currently in phase III clinical trials, the glycylcycline derivative tigecycline (GAR-936) containing a 9-tert-butylglycylamido group is part of a new generation of tetracycline antibiotics developed during the 1990s. Tigecycline displays a broad spectrum of antibacterial activity and circumvents the efflux and ribosomal protection resistance mechanisms. The TetX protein is a flavin-dependent monooxygenase that modifies first and second generation tetracyclines and requires NADPH, Mg(2+), and O(2) for activity. We report that tigecycline is a substrate for TetX and that bacterial strains containing the tet(X) gene are resistant to tigecycline. The resistance is due to the modification of tigecycline by TetX to form 11a-hydroxytigecycline, which we have shown has a weakened ability to inhibit protein translation compared with tigecycline. We have explored the basis of this decreased ability to block translation and found that hydroxylation occurs in the region of the molecule important for coordinating magnesium. 11a-Hydroxytigecycline forms a weaker complex with magnesium than tigecycline; the crystal structure of tetracycline in complex with the ribosome has shown that magnesium coordination is critical for binding tetracycline. Although tet(X) has not been isolated from any clinically resistant strains, our report demonstrates the first enzymatic resistance mechanism to tigecycline and provides an alert for the surveillance of resistant strains that may contain tet(X).