Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase

Todd Swanson, Harold B. Brooks, Andrei L. Osterman, Marion H. O'Leary, Margaret A. Phillips

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Carbon isotope effect studies were undertaken with the wild-type pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine decarboxylase (ODC) from Trypanosoma brucei and with several active site mutants of the enzyme. For the decarboxylation of the optimal substrate, L-ornithine, by wild-type ODC, the observed carbon isotope effect (k12/k13) is 1.033 at pH 7.3. In comparison to the expected intrinsic isotope effect (k12/k13 = 1.06) for decarboxylation, this value suggests that both the rate of decarboxylation and the rate of Schiff base interchange with L-ornithine are partially rate- limiting for the reaction steps up to decarboxylation. In contrast, with the alternate substrate L-Lys, which shows lower catalytic efficiency, the carbon isotope effect increased to 1.063, demonstrating that decarboxylation has become the rate-limiting step. For the mutant enzymes, E274A ODC and C360A ODC, with L-ornithine as substrate the carbon isotope effect also approaches the intrinsic limit. Glu-274 was previously demonstrated to play a direct role in carbanion stabilization, and thus the large carbon isotope effect (k12/k13 = 1.055) is consistent with an impaired rate of decarboxylation 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 than from an enzyme-bound PLP- Schiff base) has become rate-determining.

Original languageEnglish (US)
Pages (from-to)14943-14947
Number of pages5
JournalBiochemistry
Volume37
Issue number42
DOIs
StatePublished - Oct 20 1998

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase'. Together they form a unique fingerprint.

  • Cite this