TY - JOUR
T1 - Effects of proline analog binding on the spectroscopic and redox properties of PutA
AU - Zhu, Weidong
AU - Gincherman, Yekaterina
AU - Docherty, Paul
AU - Spilling, Christopher D.
AU - Becker, Donald F.
N1 - Funding Information:
We thank Dr. John J. Tanner, Department of Chemistry, University of Missouri—Columbia (Columbia, MO), for sharing information concerning the crystal structure of PutA 669 prior to publication and we acknowledge Dr. Shyamal Banerjee for help with the synthesis of 5-methylproline. This work was supported by the University of Missouri—St. Louis, Chemistry and Biochemistry Department, Research Corp. (Award RI0384); NSF (MCB0091664); NIH (GM61068); and ACS-PRF (36470-G4).
PY - 2002
Y1 - 2002
N2 - 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.
AB - 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.
KW - Competitive inhibition
KW - Lactate
KW - Proline analogs
KW - Proline dehydrogenase
KW - PutA
KW - PutA redox properties
KW - Tetrahydro-2-furoic acid
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U2 - 10.1016/S0003-9861(02)00535-0
DO - 10.1016/S0003-9861(02)00535-0
M3 - Article
C2 - 12485611
AN - SCOPUS:0036939061
VL - 408
SP - 131
EP - 136
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 1
ER -