Time-Resolved Fluorescence of O -Acetylserine Sulfhydrylase Catalytic Intermediates † (original) (raw)
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Time-resolved fluorescence of O-acetylserine sulfhydrylase
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1999
Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5P-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at V s 470 nm, indicates the presence of two components characterized by lifetimes (d) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400^560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved;
Time-resolved fluorescence of O-acetylserine sulfhydrylase1
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1999
Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5P-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at V s 470 nm, indicates the presence of two components characterized by lifetimes (d) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400^560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved;
Biophysical Journal, 2001
O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5Ј-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon twophoton excitation. We sampled the fluorescence intensity from a small number of molecules (ϳ10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity. At least two components were detected, with lifetimes of ϳ2.5 and 0.5 ns. The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state.
Conformational probes of O-acetylserine sulfhydrylase: fluorescence of tryptophans 50 and 161
Journal of Photochemistry and Photobiology B: Biology, 1999
0-acetylserine sulfhydrylase-A is a dimeric pyridoxal 5'-phosphate-dependent enzyme catalyzing the synthesis of L-cysteine. We have characterized the fluorescence properties of the two tryptophans of the enzyme, residues Trp 50 and 161, in the native state and after the binding of substrates and products, as probes of the conformational changes that take place in the apo-to holo-enzyme transformation and during the catalytic process. Upon excitation at 298 nm, the emissions of the apo-and holo-enzyme are centered at 343 and 338 nm, respectively, and are characterized by biexponential decays. The emission of the holo-enzyme is reduced by about 60% with respect to that of the apo-enzyme. The deconvolution of the peaks and the time-resolved fluorescence data indicate that (i) the emission of Trp 50 is centered at 342 and 333 nm in the apo-and holo-enzyme, respectively; (ii) the emission of Trp 161 is centered at 357 nm in both the apo-and holoenzyme; (iii) an energy transfer process quenches the fluorescence of the two tryptophan residues, being more efficient for Trp 50, which is less exposed to the solvent; (iv) the quenching of Trp 161 emission is mainly due to conformational changes accompanying the apo-to holoenzyme transition. Steady-state and time-resolved fluorescence, collected in the presence of the products acetate or L-cysteine, the product analogue L-serine, and the substrate 0-acetylserine or its analogue P-chloro-alanine, indicate that tryptophan emissions are significantly affected by variations of the equilibrium distribution between the enolimine and ketoenamine tautomers, which are differently populated during catalysis.
Rapid Photodynamics of Vitamin B 6 Coenzyme Pyridoxal 5‘-Phosphate and Its Schiff Bases in Solution
The Journal of Physical Chemistry B, 2008
The active form of vitamin B 6 , pyridoxal 5′-phosphate (PLP), is an important cofactor for numerous enzymes in amine and amino acid metabolism. Presented here is the first femtosecond transient absorption study of free PLP and two Schiff bases, PLP-valine and PLP-R-aminoisobutyric acid (AIB), in solution. Photoexcitation of free PLP leads to efficient triplet formation with an internal conversion rate that increases with increasing pH. The measured excited-state kinetics of the PLP-valine Schiff base exhibits a dramatic deuterium dependence as a result of excited-state proton transfer (ESPT) of the CR hydrogen in the amino acid substrate. This is consistent with formation of the key reaction carbanionic intermediate (quinonoid), which is resonance stabilized by the electron-deficient, conjugated π system of the Schiff base/pyridine ring. The transient absorption signals of the PLP-Schiff base with R-methylalanine (2-aminoisobutyric acid), which does not have a CR proton, lack an observable deuterium effect, verifying ESPT formation of the quinonoid intermediate. In contrast to previous studies, no dependence on the excitation wavelength of the femtosecond kinetics is observed with PLP or PLP-valine, which suggests that a rapid (<250 fs) tautomerization occurs between the enolimine (absorbing at 330 nm) and ketoenamine (absorbing at 410 nm) tautomers in solution.
Time-resolved fluorescence of tryptophan synthase
Biophysical Chemistry, 1996
Time-resolved and steady-state fluorescence of the tryptophan synthase cllpZ complex and of the pZ dimer from Sdttzotwlla typhimurium were measured to characterize the conformational properties of the p subunit in the presence and in the absence of the (Y subunit when the catalytic species internal aldimine, external aldimine and cu-aminoacrylate Schiff bases were selectively accumulated within the p active site. The fluorescence decay of the coenzyme pyridoxal 5'.phosphate, bound via a Schiff base in the p subunit of the cuZpl complex (internal aldimine species), is accounted for by two lifetimes (2.9 and 0.9 ns) of almost equal fractional intensity that are slightly affected by pH. Accordingly, both the absorption and emission spectra were found to be pH independent. The emission properties of the internal aldimine in the pz dimer are pH dependent, suggesting that the a-subunit binding alters the microenvironment of the P-subunit active site. This conclusion is also supported by the emission of the single tryptophanyl residue of the enzyme (Trp-177p). In the reaction of L-serine with the CY,~, complex, the predominant catalytic intermediate is the external aldimine (%,I,, = 422 nm) at pH 10, and the a-am%acrylate (A,,, = 350 nm) at pH 7. The external aldimine exhibits a high fluorescence intensity at 500 nm that decays with a single lifetime of 6.2 ns in the cx ?p2 complex. at pH IO. and at a similar value in the p? dimer. The emission properties of the external aldimine with respect to the internal aldimine. and the small effects induced by a-subunit binding indicate a shielding of the coenzyme and a stabilization of its excited state. In contrast. the short fluorescence lifetime (0.4 ns) and the weak fluorescence emission of the cw-aminoacrylate Schiff base indicate an increase of non-radiative processes possibly due to a more tight coupling of this intermediate with the protein matrix with respect to the external aldimine. Whereas the internal aldimine is distributed in two tautomeric forms. both the external aldimine and the cr-aminoacrylate are present in single conformational states with distinct structural and/or dynamic properties that may modulate regulatory intersubunit signals.
Journal of Molecular Structure, 2016
The results of quantum yield (QY) study of tryptophanyl glutamate (Trp-Glu), tryptophanyl lysine (Trp-Lys) and lysinyl tryptophan (Lys-Trp) dipeptides over the pH range, 1.5-13, show that the charge state of the N-terminal amine, and not the nominal molecular charge determines the QY. When the terminal amine is protonated, QY is low (10-2) for all three dipeptides. As the terminal amine cation is found proximal to the indole ring in Trp-Glu and Trp-Lys conformers but not in those for Lys-Trp, its effect may lie only in the partitioning of energy between nonradiative processes, not on QY reduction. QY is also low when both the N-terminal amine and indole amine are deprotonated. These two low QY states can be distinguished by fluorescence lifetime measurement. Molecular dynamics simulation shows that the Chi 1 conformers persist for tens of nanoseconds such that 10 0-10 1 nanosecond lifetimes may be associated with individual Chi 1 conformers. The ground state electron density or isosurface of high QY (0.30) 3-methyindole has a uniform electron density over the indole ring as do the higher QY Trp dipeptide conformers. This validates the association of ground state isosurfaces with QY. Excited state orbitals from calculated high intensity, low energy absorption transitions are typically centered over the indole ring for higher QY dipeptide species and off the ring in lower QY species. Thus excited state orbitals substantiate the earlier finding that the ground state isosurface charge density pattern on the indole ring can be predictive of QY.
Time-resolved intrinsic fluorescence of enzyme I
Biophysical Chemistry, 1992
Enzyme I of the bacterial phosphotransferase system can exist in a munomer/dimer equilibrium which may have functional significance. Each monomer contains two tryptophan residues. It is demonstrated that the decay of both the monomer and the dimer can be described by a biexponential. The decay times depend on the temperature and at 6°C the decay times are 7, = 0.4 ns and T* = 3.2 ns for the monomer and ra = 3.2 ns and r4 = 7.2 ns for the dimer form of the enzyme. The changes in the fluorescence decay parameters can be utilized to measure the equilibrium constant for the monomer/dimer transition. Keywords: Phosphotransferase system enzyme I; Time-resolved fluorescence: Monomer/dimer equilibrium 0301-4622/92/$05.00 0 1992 -Elsevier Science Publishers B.V. All rights reserved
Analysis of excited-state processes by phase-modulation fluorescence spectroscopy
Biophysical Chemistry, 1982
Fluorescence phase shift and demodulation methods were used in the analysis of excited-state reactions and to investigate solvent relaxation around fluorophores in viscous solvents. The chosen samples illustrate the results expected for fluorophores bound to biological macromolecules. These moderately simple samples served to test the theoretical predictions described in the preceding paper (J.R. Lakowicz and A.B. Balter, Biophys. Chem. 16 (1982) 99.) and to illustrate the characteristic features of phase-modulation data expected from samples which display time-dependent spectral shifts. The excited-stale protonation of acridine and exciplex formation between anthracene and diethylaniline provided examples of one-step reactions in which the lifetimes of the initially excited and the reacted species were independent of emission wavelength. Using these samples we demonstrated the following: (I) Wavelength-dependent phase shift and demodulation values can be used to prove the occurrence of an excited-state process. Proof is obtained by observation of phase angles (φ) larger than 90° or by measurement of ratios of m/cos φ > 1, where m is the modulation of the emission relative to that of the excitation. (2) For a two-state process the individual emission spectra of each state can be calculated from the wavelength-dependent phase and modulation data. (3) The phase difference or demodulation factor between the initially excited and the reacted states reveals directly the fluorescence lifetime of the product of the reaction. (4) Phase-sensitive detection of fluorescence can be used to prove that the lifetimes of both the initially excited and the reacted states are independent of emission wavelength. (5) If steady-state spectra of the individual species are known, then phase-sensitive emission spectra can be used to measure the lifetimes of the individual components irrespective of the extent of spectral overtap. (6) Spectral regions of constant lifetime can be identified by the ratios of phase-sensitive emission spectra. In addition, we examined 6-propionyl-2-dimethylaminonaphthalene(PRODAN) and N-acetyl-l-tryptophanamide (NATA) in viscous solvents where the solvent relaxation times were comparable to the fluorescence lifetimes. Using PRODAN in n-butanol we used m/cos φ measurements, relative to the blue edge of the emission, to demonstrate that solvent relaxation requires more than a single step. For NATA in propylene glycol we used phase-sensitive detection of fluorescence to directly record the emission spectra of the initially excited and the solvent relaxed states. These measurements can be easily extended to fluorophores which are bound to proteins and membranes and are likely to be useful in studies of the dynamic properties of biopolymers.