Site-directed mutagenesis provides insight into racemization and transamination of alanine catalyzed by Treponema denticola cystalysin.
In addition to alpha, beta-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Calpha/C4' of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys --> Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the k(cat)/K(m) is reduced 3.5-fold for alpha, beta-elimination, whereas it is reduced 300-400-fold for racemization. Y123F has approximately 18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face.
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
Paiardini
Alessandro
A
D'Aguanno
Simona
S
Voltattorni
Carla Borri
CB
D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D003542:Cystathionine gamma-Lyase / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011522:Protons; D013053:Spectrophotometry; D014208:Treponema / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry; D014867:Water / Q000737:chemistry
D011522:Protons; D014867:Water; D014443:Tyrosine; C400145:cystalysin; D003542:Cystathionine gamma-Lyase; D008239:Lysine; D000409:Alanine