Ornithirie decarboxylase (ODC) - a key enzyme of polyamine metabolism in C'ukaryotes. is a proven target for the development of new drugs against tr>panosomiasis. ODC cofactor - pyridoxaî-ô'-phosphate (PLP). forms a number of interactions within the active site that are essential for its tight binding and catalytic reactivity. It was previously established that Lys-69 forms a ShifT base with PLP, and Giu-274 interacts with protonated pyridinium nitrogen of PLP enhancing its electron-withdrawing capacity. In addition, modeling of ODC' structure predicts that Arg-277 forms electrostatic interactions with the phosphate moiety of PLP. We studied enzyme-cofaclor interactions in Trypanosoma brace i OUC by site-directed mutagenesis, spectral and kinetic analysis using cofactor and substrate analogs. PLP binding in the wild-type ODC was quantified by inhibition with pyridoxamine-5'-phosphate (PMP). The Kd for PMP (7 //M) is about 10-fold higher than apparent Kd for PLP. The PLP binding affinity of the K69A mutant is very similar to wild type ODC. and in the E274A mutant the apparent Kj (2 /iM) is increased only 4-fold. In both of these mutants kcat is significantly perturbed. In contrast, for the R277A mutant Kd for PLP (15 /iM) is increased almost 30-fold, but its kcat at saturating PLP is nearly the same as for wild-type ODC, supporting the predicted role of Arg-277 in PLP binding. Spectral analysis shows that several other functionally important amino acid residues, such as Lys169, Asp-361. Asp-364, directly or indirectly affect the protonation state and tautoinerism of PLP.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology