Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase.
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.
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
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T
Swanson
H B
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Brooks
A L
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Osterman
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MH
O'Leary
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Phillips
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D003653:Decarboxylation; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D009952:Ornithine / Q000737:chemistry; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012545:Schiff Bases; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D002247:Carbon Isotopes; D011993:Recombinant Fusion Proteins; D012545:Schiff Bases; D018698:Glutamic Acid; D009952:Ornithine; D009955:Ornithine Decarboxylase; D008239:Lysine