Characterization of the reaction mechanism for Trypanosoma brucei ornithine decarboxylase by multiwavelength stopped-flow spectroscopy.
Ornithine decarboxylase (ODC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the first committed step in the biosynthesis of polyamines. The UV-visible spectra of PLP (300-500 nm) was used to monitor the formation and breakdown of ODC reaction intermediates by multiwavelength stopped-flow spectroscopy to determine the reaction mechanism. Global kinetic analysis of the spectral data acquired after mixing ODC with saturating substrate (S) or product (P) (10 mM ornithine or 10 mM putrescine at 4 degrees C) suggests that ODC-catalyzed decarboxylation proceeds by the following reaction mechanism: ODC + S if A --> B --> C --> D --> E/F if ODC + P, where A-F are intermediates along the reaction path. Species B, which has absorbance maxima of 350 and 450 nm, is spectrally distinct from the other intermediates. On the basis of the calculated spectral characteristics, species B is likely to represent a quinoid intermediate which would be formed directly upon decarboxylation of ornithine. Thus, the data suggest that the reaction proceeds via formation of a Schiff base intermediate (species A) during the dead time of the stopped-flow instrument, followed by formation of a quinoid intermediate with a rate constant of 21 s-1. The quinoid intermediate decays in two steps (with rates of 145 and 1.0 s-1, respectively) to a Schiff base with putrescine (species D). Protonation of the Calpha carbon is required for the formation of species D, suggesting that the first of these events represents this step. The decay of species D to free enzyme and product occurs via a minimum of two intermediates and at an overall rate constant of 1-3 s-1. By comparison to the steady-state turnover number (kcat = 0.5 s-1 at 4 degrees C), these data identify product release as a rate-determining step in the overall reaction.
Brooks
H B
HB
Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9041, USA.
Phillips
M A
MA
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D007700:Kinetics; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D011700:Putrescine / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D009955:Ornithine Decarboxylase; D011700:Putrescine