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Release of pyridoxal 5'-phosphate upon unfolding of mitochondrial aspartate aminotransferase

Ting-Huai Wu1, Juan A Oses-Prieto, Ana Iriarte

  • 1Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Rd., Kansas City, MO 64110-2499, USA.

Biochimica Et Biophysica Acta
|April 11, 2003
PubMed

Insights

Mitochondrial aspartate aminotransferase (mAAT) unfolds in multiple steps, with pyridoxal 5'-phosphate (PLP) release occurring after catalytic inactivation, suggesting an intermediate state. This study uses PLP as a natural reporter to probe enzyme unfolding dynamics.

Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Protein structure and dynamics

Background:

  • Dimeric mitochondrial aspartate aminotransferase (mAAT) binds pyridoxal 5"-phosphate (PLP) at its active sites.
  • PLP acts as a natural reporter for studying enzyme unfolding and active site perturbations.

Purpose of the Study:

  • To investigate the multi-step unfolding process of mAAT using PLP as a reporter.
  • To determine the order of events during unfolding, including PLP release and loss of catalytic activity.

Main Methods:

  • Enzyme unfolding induced by guanidine hydrochloride (GdnHCl).
  • Monitoring PLP dissociation using size-exclusion chromatography (SEC) and electrospray mass spectrometry (ESI-MS).
  • Assessing enzyme inactivation and changes in the PLP active site environment via circular dichroism (ellipticity at 356 nm).

Main Results:

  • PLP dissociation midpoints (1.4-1.6 M GdnHCl) were higher than the inactivation midpoint (1.3 M GdnHCl).
  • The unfolding transition midpoint, reflecting the PLP active site environment, was 1.19 M GdnHCl.
  • These findings indicate that PLP remains bound after catalytic inactivation during unfolding.

Conclusions:

  • mAAT unfolding is a multi-step process.
  • An intermediate state exists during unfolding that retains bound PLP but has lost catalytic activity.
  • The study elucidates the sequential events in mAAT unfolding and the role of PLP in characterizing these transitions.

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