Spectroscopic and kinetic analyses reveal the pyridoxal 5'-phosphate binding mode and the catalytic features of Treponema denticola cystalysin.
To obtain insight into the functional properties of Treponema denticola cystalysin, we have analyzed the pH- and ligand-induced spectral transitions, the pH dependence of the kinetic parameters, and the substrate specificity of the purified enzyme. The absorption spectrum of cystalysin has maxima at 418 and 320 nm. The 320 nm band increases at high pH, while the 418 nm band decreases; the apparent pK(spec) of this spectral transition is about 8.4. Cystalysin emitted fluorescence at 367 and 504 nm upon excitation at 320 and 418 nm, respectively. The pH profile for the 367 nm emission intensity increases above a single pK of approximately 8.4. On this basis, the 418 and 320 nm absorbances have been attributed to the ketoenamine and substituted aldamine, respectively. The pH dependence of both log k(cat) and log k(cat)/K(m) for alpha,beta-elimination reaction indicates that a single ionizing group with a pK value of approximately 6.6 must be unprotonated to achieve maximum velocity. This implies that cystalysin is more catalytically competent in alkaline solution where a remarkable portion of its coenzyme exists as inactive aldamine structure. Binding of substrates or substrate analogues to the enzyme over the pH range 6-9.5 converts both the 418 and 320 nm bands into an absorbing band at 429 nm, assigned to the external aldimine in the ketoenamine form. All these data suggest that the equilibrium from the inactive aldamine form of the coenzyme shifts to the active ketoenamine form on substrate binding. In addition, reinvestigation of the substrate spectrum of alpha,beta-elimination indicates that cystalysin is a cyst(e)ine C-S lyase rather than a cysteine desulfhydrase as claimed previously.
Dipartimento di Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università degli Studi di Verona, Strada Le Grazie, 8, 37134 Verona, Italy.
Mariarita
M
Bertoldi
Barbara
B
Cellini
Tim
T
Clausen
Carla Borri
CB
Voltattorni
D002942:Circular Dichroism; D003542:Cystathionine gamma-Lyase / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014208:Treponema / Q000201:enzymology*
D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; C400145:cystalysin; D003542:Cystathionine gamma-Lyase