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B6db references: 96184911

type Journal Article
authors Ploux, O.; Marquet, A.
title Mechanistic studies on the 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis
journal Eur J Biochem
ui 96184911
year (1996)
volume 236
number 1
pages 301-8.
keywords Acyl Coenzyme A/metabolism
abstract The reaction mechanism of 8-amino-7-oxopelargonate (8-amino-7- oxononoate) synthase from Bacillus sphaericus, an enzyme dependent on pyridoxal 5'-phosphate (pyridoxal-P), which catalyzes the condensation of L-alanine with pimeloyl-CoA, the second step of biotin biosynthesis, has been studied. To facilitate mechanistic studies, an improved over- expression system in Escherichia coli, and a new continuous spectrophotometric assay for 8-amino-7-oxopelargonate synthase were designed. In order to discriminate between the two plausible basic mechanisms that can be put forth for this enzyme, that is: (a) formation of the pyridoxal-P-stabilized carbanion by abstraction of the C2-H proton of the alanine-pyridoxal-P aldimine, followed by acylation and decarboxylation, and (b) formation of the carbanion by decarboxylation followed by acylation, the fate of the C2-H proton of alanine during the course of the reaction has been examined using 1H NMR. Spectra of the 8-amino-7-oxopelargonate formed using either L-[2- 2H]alanine in H2O or L-alanine in D2O, showed that the C2-H proton of alanine is lost during the reaction and that the C8-H proton of 8-amino- 7-oxopelargonate is derived from the solvent, a result that is only consistent with mechanism (a). Furthermore 8-amino-7-oxopelargonate synthase catalyzes, in the absence of pimeloyl-CoA, the stereospecific exchange, with retention of configuration, of the C2-H proton of L- alanine with the solvent protons. Similarly, 8-amino-7-oxopelargonate synthase catalyzes the exchange of the C8-H proton of 8-amino-7- oxopelargonate. In addition to these exchange reactions, 8-amino-7- oxopelargonate synthase catalyzes an abortive transamination yielding an inactive pyridoxamine 5'-phosphate (pyridoxamine-P) form of 8-amino- 7-oxopelargonate synthase and pyruvate. Kinetic analysis gave a rate constant of kexch. = 1.8 min-1 for the exchange reaction which is 10 times lower than the catalytic constant and a rate constant of ktrans. = 0.11 h-1 for the transamination. Finally deuterium kinetic isotope effects (KIE) were measured at position 2 of L-alanine (DV = 1.3) and in D2O (D2OV = 4.0). The magnitudes of the KIE are consistent with a partially rate-limiting abstraction of the C2-H proton of alanine and a partially rate-limiting reprotonation step. Taken together, all these results show that 8-amino-7-oxopelargonate synthase utilizes mechanism (a). 8-Amino-7-oxopelargonate synthase and 5-aminolevulinate synthase, which has also been shown to use mechanism (a), belong to a class of pyridoxal-P-dependent enzymes that catalyze the formation of alpha- oxoamines. Based on the fact that all these alpha-oxoamine synthases share strong sequence similarities, we postulate that they also share the same reaction mechanism.
last changed 2002/11/12 16:17

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