Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase.

Published

Journal Article

The XL-I form of xenobiotic/medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria and used to determine the reaction mechanism. A tersubstrate kinetic analysis was conducted by varying the concentrations of ATP, benzoate and CoA in turn. Both ATP and benzoate gave parallel double-reciprocal plots against CoA, which indicates a Ping Pong mechanism, with either pyrophosphate or AMP leaving before the binding of CoA. Addition of pyrophosphate to the assays changed the plots from parallel to intersecting; addition of AMP did not. This indicates that pyrophosphate is the product that leaves before binding of CoA. Based on end-product inhibition studies, it was concluded that the reaction follows a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding first, followed in order by benzoate binding, pyrophosphate release, CoA binding, benzoyl-CoA release and AMP release. A similar mechanism was obtained when the ligase was examined with butyrate as substrate. However, butyrate activation was characterized by a much higher affinity for CoA. This is attributed to steric factors resulting from the bulkier nature of the benzoate molecule. Also, with butyrate there is a bivalent cation activation distinct from that associated with binding to ATP. This activation by excess Mg(2+) results in non-linear plots of 1/v against 1/[ATP] for butyrate unless the concentrations of Mg(2+) and ATP are varied together.

Full Text

Cited Authors

  • Vessey, DA; Kelley, M

Published Date

  • July 2001

Published In

Volume / Issue

  • 357 / Pt 1

Start / End Page

  • 283 - 288

PubMed ID

  • 11415461

Pubmed Central ID

  • 11415461

Electronic International Standard Serial Number (EISSN)

  • 1470-8728

International Standard Serial Number (ISSN)

  • 0264-6021

Digital Object Identifier (DOI)

  • 10.1042/0264-6021:3570283

Language

  • eng