title: Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications.
abstract: To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with L- and D-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429 nm and of a band absorbing at 498 nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, k (cat) and K (m), for L-alanine are 1.05+/-0.03 s(-1) and 10+/-1 mM respectively, whereas those for D-alanine are 1.4+/-0.1 s(-1) and 10+/-1 mM. During the reaction of cystalysin with L- or D-alanine, a time-dependent loss of beta-elimination activity occurs concomitantly with the conversion of the pyridoxal 5'-phosphate (PLP) coenzyme into pyridoxamine 5'-phosphate (PMP). The catalytic efficiency of the half-transamination of L-alanine is found to be 5.3x10(-5) mM(-1) x s(-1), 5-fold higher when compared with that of D-alanine. The partition ratio between racemization and half-transamination reactions is 2.3x10(3) for L-alanine and 1.4x10(4) for D-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with p K values of 6.5-6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of beta-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4' of both (4' S)- and (4' R)-[4'-(3)H]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites of L- and D-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed.
authors.affiliation: 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.
authors.forename: Mariarita
authors.initials: M
authors.lastname: Bertoldi
authors.forename: Barbara
authors.initials: B
authors.lastname: Cellini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.forename: Martino
authors.initials: M
authors.lastname: Di Salvo
authors.forename: Carla
authors.initials: C
authors.lastname: Borri Voltattorni
mesh_terms: D000409:Alanine / Q000494:pharmacology; D000411:Alanine Racemase / Q000378:metabolism*; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003542:Cystathionine gamma-Lyase / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D000637:Transaminases / Q000378:metabolism; D014208:Treponema / Q000201:enzymology*
chemical_list: D001051:Apoenzymes; D011994:Recombinant Proteins; D000637:Transaminases; C400145:cystalysin; D003542:Cystathionine gamma-Lyase; D000411:Alanine Racemase; D000409:Alanine