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Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b5 reductase for the binding of β-NADH

Paper ID Volume ID Publish Year Pages File Format Full-Text
21688 43235 2009 7 PDF Available
Title
Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b5 reductase for the binding of β-NADH
Abstract

Well-conserved three consecutive Pro residues (Pro247–249) in the NADH-binding subdomain of NADH-cytochrome b5 reductase were proposed to form a basal part of the NADH-binding site. To investigate the structural and mechanistic roles of these residues, we expressed site-directed mutants for a soluble domain of the porcine enzyme where each of the residues was replaced with either Ala or Leu residue, respectively, using a heterologous expression system in Escherichia coli. Six mutants (P247A, P247L, P248A, P248L, P249A, and P249L) were produced as a fusion protein containing a 6×His-tag sequence at the NH2-terminus and were purified to homogeneity with a stoichiometric amount of bound FAD. Mutations were each confirmed for the purified proteins by MALDI-TOF mass spectrometry. Steady-state kinetic analyses for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase acitivities were conducted for all the mutants. Substitution of Pro247 with Leu residue was found to significantly decrease kcat with slight increase in Km for the physiological electron donor NADH. However, Km values for the electron acceptors (both cytochrome b5 and ferricyanide) of P247L were found to be decreased significantly. Such changes were not observed for P247A or other four mutants. These results suggested that Pro247 among the three consecutive Pro residues has the most important role for the formation of a binding site cavity and that only a slight change in the side-chain volume at this residue from Ala to Leu residue affected the electron transfer reaction from NADH and, further, on the recognition of ferricytochrome b5.

Keywords
NADH binding; Electron transfer; Site-directed mutagenesis; Pro residue; FAD
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Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b5 reductase for the binding of β-NADH
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 108, Issue 4, October 2009, Pages 286–292
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us