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 Citations (Scopus)

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 Δ 1 -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 K i = 0.2 mM) and to perturb the flavin adenine dinucleotide (FAD) absorbance spectrum upon complexation to PutA. At pH 7.5, a reduction potential (E m ) of -0.089 V for the FAD/FADH 2 couple in L-THFA-complexed PutA was determined by potentiometric titrations. The E m value for L-THFA-complexed PutA is 12 mV more negative than the E m for uncomplexed PutA (E m = -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
StatePublished - Jan 1 2002

Fingerprint

Proline
Flavin-Adenine Dinucleotide
Oxidation-Reduction
Proline Oxidase
1-Pyrroline-5-Carboxylate Dehydrogenase
Flavoproteins
Regulon
Transcription
Complexation
Titration
Metabolism
Escherichia coli
Glutamic Acid
Cytoplasm
Membranes
2-furoic acid
Testing
Substrates

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Effects of proline analog binding on the spectroscopic and redox properties of PutA. / Zhu, Weidong; Gincherman, Yekaterina; Docherty, Paul; Spilling, Christopher D.; Becker, Donald F.

In: Archives of Biochemistry and Biophysics, Vol. 408, No. 1, 01.01.2002, p. 131-136.

Research output: Contribution to journalArticle

Zhu, Weidong ; Gincherman, Yekaterina ; Docherty, Paul ; Spilling, Christopher D. ; Becker, Donald F. / Effects of proline analog binding on the spectroscopic and redox properties of PutA. In: Archives of Biochemistry and Biophysics. 2002 ; Vol. 408, No. 1. pp. 131-136.
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