Lysine 238 is an essential residue for alpha,beta-elimination catalyzed by Treponema denticola cystalysin.
Treponema denticola cystalysin is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the alpha,beta-elimination of l-cysteine to pyruvate, ammonia, and H2S. Similar to other PLP enzymes, an active site Lys residue (Lys-238) forms an internal Schiff base with PLP. The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R). Both apomutants reconstituted with PLP bind noncovalently approximately 50% of the normal complement of the cofactor and have a lower affinity for the coenzyme than that of wild-type. Kinetic analyses of the reactions of K238A and K238R mutants with glycine compared with that of wild-type demonstrate the decrease of the rate of Schiff base formation by 103- and 7.5 x 104-fold, respectively, and, to a lesser extent, a decrease of the rate of Schiff base hydrolysis. Thus, a role of Lys-238 is to facilitate formation of external aldimine by transimination. Kinetic data reveal that the K238A mutant is inactive in the alpha,beta-elimination of l-cysteine and beta-chloro-l-alanine, whereas K238R retains 0.3% of the wild-type activity. These data, together with those derived from a spectral analysis of the reaction of Lys-238 mutants with unproductive substrate analogues, indicate that Lys-238 is an essential catalytic residue, possibly participating as a general base abstracting the Calpha-proton from the substrate and possibly as a general acid protonating the beta-leaving group.
Bertoldi
Mariarita
M
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.
Cellini
Barbara
B
D'Aguanno
Simona
S
Borri Voltattorni
Carla
C
D002384:Catalysis; D002942:Circular Dichroism; D003542:Cystathionine gamma-Lyase / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine; D011732:Pyridoxal Phosphate / Q000378:metabolism; D014208:Treponema / Q000201:enzymology*
D011732:Pyridoxal Phosphate; C400145:cystalysin; D003542:Cystathionine gamma-Lyase; D008239:Lysine