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 ornithine decarboxylase (ODC) has been studied by site-directed mutagenesis and spectroscopy. The beta/alpha barrel model proposed for the eukaryotic ODC structure predicts that the phosphate group of PLP is stabilized 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 ODC while reducing k(cat) by only 2-fold at pH 8. PLP binding affinity was measured directly by ultrafiltration; the K(d) for PLP is at least 20-fold higher in the mutant enzyme at pH 8. In addition, R277A ODC also has weaker binding affinities for a series of cofactor analogs than the wild-type enzyme. These results demonstrate that Arg-277 is necessary for high-affinity PLP binding by ODC. The 31P NMR spectra of ODC suggest that the phosphate is bound in a strained conformation as a dianion to both wild-type and R277A ODC. However, the 31P chemical shift 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-bound phosphate is altered in the mutant enzyme. The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, corresponding to the titration of a protonated species with a pK(a) of approximately 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 31P chemical shift and a CD signal observable over the entire tested pH range (7-9). In contrast, for R277A ODC between pH 8 and 9, the 31P 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 essential for efficient catalysis, tempering the consequence of the removal of any single interaction.
Osterman
A L
AL
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Brooks
H B
HB
Rizo
J
J
Phillips
M A
MA
D000818:Animals; D001120:Arginine / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D011733:Pyridoxamine / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014462:Ultrafiltration
D065108:Ornithine Decarboxylase Inhibitors; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; C002064:4-deoxypyridoxine 5'-phosphate; D001120:Arginine; D009955:Ornithine Decarboxylase