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Start Over Author "C. Blouin" Journal biochemistry
3 results on '"C. Blouin"'

1. Straight-Chain Alkyl Isocyanides Open the Distal Histidine Gate in Crystal Structures of Myoglobin

Author

George C. Blouin, John S. Olson, George N. Phillips, Kenneth A. Johnson, and Robert D. Smith

Subjects
Models, Molecular, chemistry.chemical_classification, education.field_of_study, Cyanides, Myoglobin, Protein Conformation, Stereochemistry, Ligand, Population, Crystal structure, Hydrogen-Ion Concentration, Crystallography, X-Ray, Ligands, Biochemistry, Article, chemistry.chemical_compound, chemistry, Nitriles, Side chain, Histidine, education, Conformational isomerism, Alkyl
Abstract

Crystal structures of methyl, ethyl, propyl, and butyl isocyanide bound to sperm whale myoglobin (Mb) reveal two major conformations. In the in conformer, His(E7) is in a "closed" position, forcing the ligand alkyl chain to point inward. In the out conformer, His(E7) is in an "open" position, allowing the ligand side chain to point outward. A progressive increase in the population of the out conformer is observed with increasing ligand length in P2(1) crystals of native Mb at pH 7.0. This switch from in to out with increasing ligand size also occurs in solution as measured by the decrease in the relative intensity of the low-frequency ( approximately 2075 cm(-1)) versus high-frequency ( approximately 2125 cm(-1)) isocyano bands. In contrast, all four isocyanides in P6 crystals of wild-type recombinant Mb occupy the in conformation. However, mutating either His64 to Ala, creating a "hole" to solvent, or Phe46 to Val, freeing rotation of His64, causes bound butyl isocyanide to point completely outward in P6 crystals. Thus, the unfavorable hindrance caused with crowding a large alkyl side chain into the distal pocket appears to be roughly equal to that for pushing open the His(E7) gate and is easily affected by crystal packing. This structural conclusion supports the "side path" kinetic mechanism for O(2) release, in which the dissociated ligand first moves toward the protein interior and then encounters steric resistance, which is roughly equal to that for escaping to solvent through the His(E7) channel.

Published
2010
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2. Tyrosine B10 Inhibits Stabilization of Bound Carbon Monoxide and Oxygen in Soybean Leghemoglobin

Author

Scott A. Premer, Suman Kundu, George C. Blouin, John S. Olson, Gautam Sarath, and Mark S. Hargrove

Subjects
Models, Molecular, Stereochemistry, Inorganic chemistry, chemistry.chemical_element, Ligands, Biochemistry, Oxygen, chemistry.chemical_compound, Spectroscopy, Fourier Transform Infrared, Side chain, Animals, Histidine, Leghemoglobin, Carbon Monoxide, Myoglobin, Hydrogen bond, Imidazoles, Whales, Oxygen transport, food and beverages, Hydrogen Bonding, chemistry, Mutagenesis, Site-Directed, Tyrosine, Soybeans, Protein Binding, Carbon monoxide
Abstract

Detailed comparisons of the carbon monoxide FTIR spectra and ligand-binding properties of a library of E7, E11, and B 10 mutants indicate significant differences in the role of electrostatic interactions in the distal pockets of wild-type sperm whale myoglobin and soybean leghemoglobin. In myoglobin, strong hydrogen bonds from several closely related conformations of the distal histidine (His E 7 ) side chain preferentially stabilize bound oxygen. In leghemoglobin, the imidazole side chain of His E 7 is confined to a single conformation, which only weakly hydrogen bonds to bound ligands. The phenol side chain of Tyr B 1 0 appears to "fix" the position of His E 7 , probably by donating a hydrogen bond to the Nδ atom of the imidazole side chain. The proximal pocket of leghemoglobin is designed to favor strong coordination bonds between the heme iron and axial ligands. Thus, high oxygen affinity in leghemoglobin is established by a favorable staggered geometry of the proximal histidine. The interaction between His E 7 and Tyr B 1 0 prevents overstabilization of bound oxygen. If hydrogen bonding from His E 7 were as strong as it is in mammalian myoglobin, the resultant ultrahigh affinity of leghemoglobin would prevent oxygen transport in root nodules.

Published
2004
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3. Perturbing the hydrophobic pocket of mandelate racemase to probe phenyl motion during catalysis.

Author

Siddiqi F, Bourque JR, Jiang H, Gardner M, St Maurice M, Blouin C, and Bearne SL

Subjects
Binding Sites, Catalysis, Computer Simulation, Hydrophobic and Hydrophilic Interactions, Hydroxybutyrates chemistry, Hydroxybutyrates pharmacology, Isomerism, Kinetics, Mandelic Acids chemistry, Mandelic Acids metabolism, Models, Molecular, Mutation genetics, Phenylalanine genetics, Phenylalanine metabolism, Protein Structure, Tertiary, Pseudomonas putida enzymology, Pseudomonas putida genetics, Racemases and Epimerases genetics, Movement, Phenol chemistry, Phenol metabolism, Racemases and Epimerases chemistry, Racemases and Epimerases metabolism
Abstract

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Crystal structures of MR reveal that the phenyl group of all ground-state ligands is located within a hydrophobic cavity, remote from the site of proton abstraction. MR forms numerous electrostatic and H-bonding interactions with the alpha-OH and carboxyl groups of the substrate, suggesting that these polar groups may remain relatively fixed in position during catalysis while the phenyl group is free to move between two binding sites [i.e., the R-pocket and the S-pocket for binding the phenyl group of (R)-mandelate and (S)-mandelate, respectively]. We show that MR binds benzilate (K(i) = 0.67 +/- 0.12 mM) and (S)-cyclohexylphenylglycolate (K(i) = 0.50 +/- 0.03 mM) as competitive inhibitors with affinities similar to that which the enzyme exhibits for the substrate. Therefore, the active site can simultaneously accommodate two phenyl groups, consistent with the existence of an R-pocket and an S-pocket. Wild-type MR exhibits a slightly higher affinity for (S)-mandelate [i.e., K(m)(S)(-)(man) < K(m)(R)(-)(man)] but catalyzes the turnover of (R)-mandelate slightly more rapidly (i.e., k(cat)(R)(-->)(S) > k(cat)(S)(-->)(R)). Upon introduction of steric bulk into the S-pocket using site-directed mutagenesis (i.e., the F52W, Y54W, and F52W/Y54W mutants), this catalytic preference is reversed. Although the catalytic efficiency (k(cat)/K(m)) of all the mutants was reduced (11-280-fold), all mutants exhibited a higher affinity for (R)-mandelate than for (S)-mandelate, and higher turnover numbers with (S)-mandelate as the substrate, relative to those with (R)-mandelate. (R)- and (S)-2-hydroxybutyrate are expected to be less sensitive to the additional steric bulk in the S-pocket. Unlike those for mandelate, the relative binding affinities for these substrate analogues are not reversed. These results are consistent with steric obstruction in the S-pocket and support the hypothesis that the phenyl group of the substrate may move between an R-pocket and an S-pocket during racemization. These conclusions were also supported by modeling of the binary complexes of the wild-type and F52W/Y54W enzymes with the substrate analogues (R)- and (S)-atrolactate, and of wild-type MR with bound benzilate using molecular dynamics simulations.

Published
2005
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