Effects of proline analog binding on the spectroscopic and redox properties of PutA

Weidong Zhu, Yekaterina Gincherman, Paul Docherty, Christopher D. Spilling, Donald F. Becker

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

The PutA flavoprotein regulates proline metabolism in Escherichia coli by performing two distinct functions. First, in the cytoplasm, PutA represses transcription of the put (proline utilization) regulon. Second, PutA associates with the membrane to oxidize proline to glutamate using discrete proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase domains. Here, we identify a proline analog that will be useful for testing the role substrate binding has in regulating PutA functions. L-Tetrahydro-2-furoic acid (L-THFA) was found to display simple competitive inhibition of proline dehydrogenase activity in PutA (apparent Ki = 0.2 mM) and to perturb the flavin adenine dinucleotide (FAD) absorbance spectrum upon complexation to PutA. At pH 7.5, a reduction potential (Em) of -0.089 V for the FAD/FADH2 couple in L-THFA-complexed PutA was determined by potentiometric titrations. The Em value for L-THFA-complexed PutA is 12 mV more negative than the Em for uncomplexed PutA (Em = -0.077 V, pH 7.5) and corresponds to just a twofold increase in the dissociation constant of L-THFA with PutA upon reduction of FAD.

Original languageEnglish (US)
Pages (from-to)131-136
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume408
Issue number1
DOIs
Publication statusPublished - Jan 1 2002

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Keywords

  • Competitive inhibition
  • Lactate
  • Proline analogs
  • Proline dehydrogenase
  • PutA
  • PutA redox properties
  • Tetrahydro-2-furoic acid

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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