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, kcat and Km, 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 β-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 5.3 × 10-5 mM-1 · s-1, 5-fold higher when compared with that of D-alanine. The partition ratio between racemization and half-transamination reactions is 2.3 × 103 for L-alanine and 1.4 × 104 for D-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with pK 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 β-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′-3H]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.
Treponema denticola cystalysin exhibits a significant alanine racemase activity accompanied by transamination: mechanistic implications
BERTOLDI, Mariarita;CELLINI, Barbara;VOLTATTORNI, Carla
2003-01-01
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, kcat and Km, 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 β-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 5.3 × 10-5 mM-1 · s-1, 5-fold higher when compared with that of D-alanine. The partition ratio between racemization and half-transamination reactions is 2.3 × 103 for L-alanine and 1.4 × 104 for D-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with pK 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 β-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′-3H]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.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
