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Molecular basis of the medium-chain fatty acyl-CoA dehydrogenase-catalyzed "oxidase" reaction: pH-dependent distribution of intermediary enzyme species during catalysis.

Biochemistry (1994-04-19)
J K Johnson, N R Kumar, D K Srivastava
ABSTRACT

In a previous paper, we demonstrated that the medium-chain fatty acyl-CoA dehydrogenase-catalyzed (MCAD-catalyzed) reductive half-reaction of indolepropionyl-CoA proceeds via formation of a chromophoric intermediary species "X" (absorption maximum = 400 nm) and proposed that the decay of this species might limit the overall rate of the "oxidase" reaction [Johnson, J. K., & Srivastava, D. K. (1993) Biochemistry 32, 8004-8013]. During this latter reaction, the buffer-dissolved O2 served as an electron acceptor [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. To ascertain whether the intrinsic stability of X influences the oxidase activity, we undertook a detailed kinetic investigation of this enzyme at different pH values. The time-resolved spectra for the reductive half-reaction (obtained via the rapid-scanning stopped-flow method) at different pH values reveal that the amplitude of the intermediary (X) spectral band is more pronounced at a lower pH (pH 6.4) than at a higher pH (pH 9.0). Single-wavelength transient kinetic data for the reductive half-reaction (in both the forward and the reverse direction) at all pH values are consistent with fast (1/tau 1) and slow (1/tau 2) relaxation rate constants. Of these, whereas the fast relaxation rate constant for the reaction in the forward direction (1/tau 1f) decreases with an increase in pH, the corresponding slow relaxation rate constant (1/tau 2f) increases with an increase in pH. The pH-dependent steady-state kinetic data reveal that, like 1/tau 2f, kcat for the MCAD-catalyzed oxidase reaction increases with an increase in the pH of the buffer media.(ABSTRACT TRUNCATED AT 250 WORDS)

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3-Indoleacrylic acid, BioReagent, ≥98% (HPLC)
Sigma-Aldrich
trans-3-Indoleacrylic acid, 98%
Sigma-Aldrich
3-Indoleacrylic acid, powder

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