TIME-RESOLVED FLUORESCENCE OF O-ACETYLSERINE SULFHYDRYLASE CATALYTIC INTERMEDIATES

The reaction of the substrate O-acetyl-L-serine (GAS) with the pyridox al 5'-phosphate (PLP)dependent enzyme O-acetylserine sulfhydrylase-A ( OASS-A) proceeds via the transient formation of an external aldimine a bsorbing at 420 nm and a stable alpha-aminoacrylate intermediate absor bing at 330 and 465 nm. Stable external aldimine species are obtained by reaction of the enzyme with either the reaction product L-cysteine or the product analog L-serine. Static and time-resolved fluorescence emission properties of the coenzyme in the above catalytic intermediat es have been used to directly probe the active site conformation at di fferent stages of the catalytic pathway. Upon excitation at either 420 or 330 nm, the external aldimines with L-cysteine and L-serine exhibi t a structured emission centered at 490 nm with a shoulder at 530 nm. Fluorescence decays upon excitation at 420 nm are best fitted using tw o components with lifetimes of 1.1 and 3.8 ns, with the fractional int ensity of the slow component being 0.92 with L-cysteine and 0.75 with L-serine, respectively. The fast component, emitting at 530 nm, is att ributed to a dipolar species formed in the excited state by proton dis sociation, and the slow component, emitting at 490 nm, is attributed t o a ketoenamine tautomer of the external aldimine. The slow component for external aldimine fluorescence decay is characterized by the same lifetime value as that of the internal aldimine with an increased frac tional intensity, indicating that the distribution between the ketoena mine and the dipolar species is shifted toward the ketoenamine tautome r in the external aldimine, compared to the internal aldimine. Differe nces in equilibrium distribution of ketoenamine and enolimine tautomer s can also account for differences in the emission properties of the e xternal aldimines of L-cysteine and L-serine. The alpha-aminoacrylate species is characterized by a relatively weak emission. Upon excitatio n at 330 nm, the emission exhibits two bands centered at 420 and 540 n m, whereas upon excitation at 420 nm the emission bands are centered a t 500 and 540 nm, and upon excitation at 465 nm, the main absorbance p eak of the alpha-aminoacrylate species, the emission spectrum shows a band at 540 nm. The fluorescence decays, upon excitation at 330 nm, ar e best fitted using three components with lifetime values similar to t hose found for the internal aldimine, with the slow component predomin ating. Species-associated spectra, collected between 400 and 520 nm up on excitation at 350 nm, indicate the presence of a fast component ove rlapping the slow component on the blue side of the emission spectrum, as detected for the internal aldimine. When the excitation wavelength is 420 nm, there are only two components with the fast one predominat ing. A further increase in the fractional intensity of the fast compon ent is observed upon excitation at 465 nm. The weak emission and the s hort lifetime of the emission excited at 465 nm indicate that this alp ha-aminoacrylate tautomer interacts significantly with neighboring gro ups of the protein matrix and may be endowed with a higher mobility th an the external aldimine.