TY - JOUR
T1 - Lysine 238 is an essential residue for α,β-elimination catalyzed by Treponema denticola cystalysin
AU - Bertoldi, Mariarita
AU - Cellini, Barbara
AU - D'Aguanno, Simona
AU - Voltattorni, Carla Borri
PY - 2003/9/26
Y1 - 2003/9/26
N2 - Treponema denticola cystalysin is a pyridoxal 5′-phosphate (PLP) enzyme that catalyzes the α,β-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 The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R).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 ∼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 × 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 α,β-elimination of L-cysteine and β-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 Cα-proton from the substrate and possibly as a general acid protonating the β-leaving group.
AB - Treponema denticola cystalysin is a pyridoxal 5′-phosphate (PLP) enzyme that catalyzes the α,β-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 The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R).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 ∼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 × 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 α,β-elimination of L-cysteine and β-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 Cα-proton from the substrate and possibly as a general acid protonating the β-leaving group.
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U2 - 10.1074/jbc.M305967200
DO - 10.1074/jbc.M305967200
M3 - Article
C2 - 12882978
AN - SCOPUS:0141844593
VL - 278
SP - 37336
EP - 37343
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 39
ER -