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 Citations (Scopus)

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

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Carbon Isotopes
Trypanosoma brucei brucei
Decarboxylation
Ornithine Decarboxylase
Isotopes
Carbon
Pyridoxal Phosphate
Ornithine
Schiff Bases
Enzymes
Substrates
Interchanges
Catalytic Domain
Stabilization

ASJC Scopus subject areas

  • Biochemistry

Cite this

Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase. / Swanson, Todd; Brooks, Harold B.; Osterman, Andrei L.; O'Leary, Marion H.; Phillips, Margaret A.

In: Biochemistry, Vol. 37, No. 42, 20.10.1998, p. 14943-14947.

Research output: Contribution to journalArticle

Swanson, T, Brooks, HB, Osterman, AL, O'Leary, MH & Phillips, MA 1998, 'Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase', Biochemistry, vol. 37, no. 42, pp. 14943-14947. https://doi.org/10.1021/bi981154i
Swanson, Todd ; Brooks, Harold B. ; Osterman, Andrei L. ; O'Leary, Marion H. ; Phillips, Margaret A. / Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase. In: Biochemistry. 1998 ; Vol. 37, No. 42. pp. 14943-14947.
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