Altering the reaction specificity of eukaryotic ornithine decarboxylase

Ornithine decarboxylase (ODC) catalyzes the first committed step in the bio synthesis of polyamines, and it has been identified as a drug target for th e treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodim er, X-ray structural analysis of the complex of the T. brucei wild-type enz yme with the product putrescine reveals two structural changes that occur u pon ligand binding: Lys-69 is displaced by putrescine and forms new interac tions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into th e active site to within 3.4 Angstrom of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- an d 1000-fold, respectively. However, HPLC analysis of the products demonstra tes that the mutant enzymes almost exclusively catalyze a decarboxylation-d ependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reactio n arises when the decarboxylated substrate intermediate is protonated at C4 ' of PLP instead of at the C-alpha of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (<0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' o ccurs either very rapidly or in a concerted reaction with decarboxylation a nd that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C-alpha protonation step that catalyzes the formation o f the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls r eaction specificity.