Probing the role of Tyr 64 of Treponema denticola cystalysin by site-directed mutagenesis and kinetic studies.
Tyr 64, hydrogen-bonded to coenzyme phosphate in Treponema denticola cystalysin, was changed to alanine by site-directed mutagenesis. Spectroscopic and kinetic properties of the Tyr 64 mutant were investigated in an effort to explore the differences in coenzyme structure and kinetic mechanism relative to those of the wild-type enzyme. The wild type displays coenzyme absorbance bands at 418 and 320 nm, previously attributed to ketoenamine and substituted aldamine, respectively. The Tyr 64 mutant exhibits absorption maxima at 412 and 325 nm. However, the fluorescence characteristics of the latter band are consistent with its assignment to the enolimine form of the Schiff base. pK(spec) values of approximately 8.3 and approximately 6.5 were observed in a pH titration of the wild-type and mutant coenzyme absorbances, respectively. Thus, Tyr 64 is probably the residue involved in the nucleophilic attack on C4' of pyridoxal 5'-phosphate (PLP) in the internal aldimine. Although the Tyr 64 mutant exhibits a lower affinity for PLP and lower turnover numbers for alpha,beta-elimination and racemization than the wild type, the pH profiles for their Kd(PLP) and kinetic parameters are very similar. Rapid scanning stopped-flow and chemical quench experiments suggest that, in contrast to the wild type, for which the rate-determining step of alpha,beta-elimination of beta-chloro-L-alanine is the release of pyruvate, the rate-determining step for the mutant in the same reaction is the formation of alpha-aminoacrylate. Altogether, these results provide new insights into the catalytic mechanism of cystalysin and highlight the functional role of Tyr 64.
Cellini
Barbara
B
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.
Bertoldi
Mariarita
M
Montioli
Riccardo
R
Borri Voltattorni
Carla
C
D000818:Animals; D001483:Base Sequence; D003542:Cystathionine gamma-Lyase / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000737:chemistry; D048373:Treponema denticola / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D014443:Tyrosine; D011732:Pyridoxal Phosphate; C400145:cystalysin; D003542:Cystathionine gamma-Lyase