ROLE OF ARG-277 IN THE BINDING OF PYRIDOXAL 5'-PHOSPHATE TO TRYPANOSOMA-BRUCEI ORNITHINE DECARBOXYLASE

The pyridoxal 5'-phosphate (PLP) binding site in Trypanosoma brucei or nithine decarboxylase (ODC) has been studied by site-directed mutagene sis and spectroscopy. The beta/alpha barrel model proposed for the euk aryotic ODC structure predicts that the phosphate group of PLP is stab ilized by interactions with a Gly-rich loop (residues 235-237) and by a salt bridge to Arg-277 [Grishin, N. V., Phillips, M. A., & Goldsmith , E. J. (1995) Protein Sci. 4, 1291-1304]. Mutation of Arg-277 to Ala increases the K-m for PLP by 270-fold compared to that of wild-type OD C while reducing k(cat) by only 2-fold at pH 8. PLP binding affinity w as measured directly by ultrafiltration; the K-d for PLP is at least 2 0-fold higher in the mutant enzyme at pH 8. In addition, R277A ODC als o has weaker binding affinities for a series of cofactor analogs than the wild-type enzyme. These results demonstrate that Arg-277 is necess ary for high-affinity PLP binding by ODC. The P-31 NMR spectra of ODC suggest that the phosphate is bound in a strained conformation as a di anion to both wild-type and R277A ODC. However, the P-31 chemical shif t for R277A ODC (6.7 ppm) is 0.5 ppm downfield from that observed for the wild-type enzyme, indicating that the environment of the enzyme-bo und phosphate is altered in the mutant enzyme. The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, correspond ing to the titration of a protonated species with a pK(a) of approxima tely 8.5. Concomitant with these changes are a decreased k(cat) and an altered absorption spectra which arises from bound PLP. PLP bound to wild-type ODC has a P-31 chemical shift and a CD signal observable ove r the entire tested pH range (7-9). In contrast, for R277A ODC between pH 8 and 9, the P-31 chemical shift becomes solution-like and the CD signal is abolished. The data suggest that for R277A ODC the rigid PLP binding mode which characterizes the wild-type enzyme is lost at high pH. Thus, multiple interactions between the wild-type active site and PLP maintain the cofactor in a constrained conformation that is essen tial for efficient catalysis, tempering the consequence of the removal of any single interaction.