C-13 ISOTOPE EFFECT STUDIES OF TRYPANOSOMA-BRUCEI ORNITHINE DECARBOXYLASE

Carbon isotope effect studies were undertaken with the wild-type pyrid oxal 5'-phosphate (PLP)dependent enzyme ornithine decarboxylase (ODC) from Trypanosoma brucei and with several active site mutants of the en zyme. For the decarboxylation of the optimal substrate, L-ornithine, b y wild-type ODC, the observed carbon isotope effect (k(12)/k(13)) is 1 .033 at pH 7.3. In comparison to the expected intrinsic isotope effect (k(12)/k(13) = 1.06) for decarboxylation, this value suggests that bo th the rate of decarboxylation and the rate of Schiff base interchange with L-ornithine are partially rate-limiting for the reaction steps u p to decarboxylation. In contrast, with the alternate substrate L-Lys, which shows lower catalytic efficiency, the carbon isotope effect inc reased to 1.063, demonstrating that decarboxylation has become the rat e limiting step. For the mutant enzymes, E274A ODC and C360A ODC, with L-ornithine as substrate the carbon isotope effect also approaches th e intrinsic limit. Glu-274 was previously demonstrated to play a direc t role in carbanion stabilization, and thus the large carbon isotope e ffect (k(12)/k(13) = 1.055) is consistent with an impaired rate of dec arboxylation compared to wild-type ODC. In contrast, for K69A ODC, the isotope effect is almost entirely suppressed, suggesting that Schiff- base formation (which now must occur from enzyme-bound PLP, rather tha n from an enzyme-bound PLP-Schiff base) has become rate-determining.