Figure 6
Results of the Schiff base treatments (reduction to non-hydrolyzable secondary amine and conversion to non-covalently bound PMP) on the spectroscopic and activity profile of the two catalytically active wild-type (WT) and Y322A Cv-ATA. For both enzymes, the modification of the Schiff base using either of the two methods resulted in the red-shift of the absorption maximum characteristic of the Schiff base (~420 nm) (panels a and b, referring to the Cv-ATA WT and Y322A, respectively). For both enzymes the conversion of the cofactor PLP to non-covalently bound PMP at room temperature in the absence of amino donors resulted in the rapid decrease in activity of both Cv-ATA enzyme variants, WT and Y322A (panel c). The incubation in the presence of either amino donor or reducing agent (4 h, on ice) show that for both enzymes the catalytic activity is irreversibly lost (panel d). While the reduction of the Schiff base completely obliterates the enzymatic activity, the conversion to PMP in the absence of an amino acceptor reduces the enzymatic activity to 44.4% and 31.6% for the Cv-ATA WT and Y322A, respectively.
