Cytochrome b 5 (cyt b 5 ) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane ␣-helix at its carboxyl terminus. These two domains are connected by an ϳ15-amino acid linker domain, Ser 90 -Asp 104 , which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b 5 to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b 5 to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b 5 except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b 5 to interact with cyt P450 2B4. A model for the membranebound cyt b 5 -cyt P450 complex is presented.Microsomal cytochrome b 5 (cyt b 5 ) 1 is an amphipathic electron transfer hemoprotein located in the membrane of the endoplasmic reticulum. It provides electrons for a broad range of reactions, including fatty acid desaturation, cholesterol biosynthesis, and a variety of cytochrome P450-dependent oxidation and hydroxylation reactions (1, 2).Cytochrome P450 (cyt P450) is responsible for the oxidation of a large number of substrates. The overall stoichiometry of the reaction catalyzed by cyt P450 is shown in Reaction 1, where RH is the substrate (3).The cyt P450 catalytic reaction occurs via a reaction cycle that involves substrate binding, reduction of the ferric heme, O 2 binding, reduction of the oxyferrous heme (second electron transfer), substrate oxidation, and finally product dissociation (4). Both ferric and oxyferrous cyt P450 receive electrons from NADPH via its redox partner, NADPH-cytochrome P450 reductase (cyt P450 reductase), whereas cyt b 5 can donate the second electron. Because of the high redox potential of Х20 mV for cyt b 5 , the first electron required to reduce cyt P450 from the ferric to the ferrous state is always transferred from NADPH via cyt P450 reductase. However, as the redox potential of cyt P450 increases from ХϪ230 mV to ϩ50 mV on transition to the oxyferrous state, the second electron can be obtained from either cyt P450 reductase or cyt b 5 (5, 6).Depending on the substrate, isozyme of cyt P450 and the experimental conditions cyt b 5 can stimulate, inhibit, or have no effect on cyt P450 activity (2). Studies of the stoichiometry of the metabolism of benzphetamine and methoxyflurane by cyt P450 2B4 have shown that cyt b 5 increases the efficiency of catalysis by cyt P450 2B4 primarily by decreasing the formation of the side-product superoxide. However, cyt b...

Cyanide binding to a cytochrome c peroxidase (CcP) variant in which the distal histidine has been replaced by a leucine residue, CcP(H52L), has been investigated as a function of pH using spectroscopic, equilibrium, and kinetic methods. Between pH 4 and 8, the apparent equilibrium dissociation constant for the CcP(H52L)/cyanide complex varies by a factor of 60, from 135 microM at pH 4.7 to 2.2 microM at pH 8.0. The binding kinetics are biphasic, involving bimolecular association of the two reactants, followed by an isomerization of the enzyme/cyanide complex. The association rate constant could be determined up to pH 8.9 using pH-jump techniques. The association rate constant increases by almost 4 orders of magnitude over the pH range investigated, from 1.8 x 10(2) M(-1) s(-1) at pH 4 to 9.2 x 10(5) M(-1) s(-1) at pH 8.6. In contrast to wild-type CcP, where the binding of HCN is the dominant binding pathway, CcP(H52L) preferentially binds the cyanide anion. Above pH 8, cyanide binding to CcP(H52L) is faster than cyanide binding to wild-type CcP. Cyanide dissociates 4 times slower from the mutant protein although the pH dependence of the dissociation rate constant is essentially identical for CcP(H52L) and CcP. Isomerization of the CcP(H52L)/cyanide complex is observed between pH 4 and 8 and stabilizes the complex. The isomerization rate constant has a similar magnitude and pH dependence as the cyanide dissociation rate constant, and the two reactions are coupled at low cyanide concentrations. This isomerization has no counterpart in the wild-type CcP/cyanide complex.

Interaction of curcumin (CUR) with the enzyme dihydrofolate reductase (DHFR) was studied by molecular docking using AutoDock 4.2 as the docking software application. AutoDock 4.2 software serves as a valid and acceptable docking application to study the interactions of small compounds with proteins. Interactions of curcumin with DHFR were compared to those of methotrexate (MTX), a known inhibitor of the enzyme. The calculated free energy of binding (ΔG binding) shows that curcumin (ΔG = -9.02 kcal/mol; Ki = 243 nM) binds with affinity comparable to or better than MTX (ΔG = -8.78 kcal/mol; Ki = 363 nM). Binding interactions of curcumin with active site residues of the enzyme are also predicted. Curcumin appears to bind in a bent conformation making extensive VDW contacts in the active site of the enzyme. Hydrogen bonding and pi-pi interaction with key active site residues are also observed. Thus, curcumin can be considered as a good lead compound in the development of new inhibitors of DHFR, which is a potential target of anti-cancer drugs. The results of these studies can serve as a starting point for further computational and experimental studies.

Psoriasis is one of the most prevalent chronic inflammatory diseases of the skin. The Wnt pathways have been documented to play essential role in stem cell self-renewal and keratinocyte differentiation in the skin. Antagonizing the Wnt5a protein would emerge as a novel therapeutics in psoriasis treatment. In this view, we have developed and characterized series of compounds by attaching varied tertiary alkyloxy carbonyl groups at the N-terminal end of the hexapeptide (Met-Asp-Gly-Cys-Glu-Leu) bestowed to inhibit Wnt/Ca2+ signaling in psoriasis. Hexapeptide compound with 1,1-diphenylethoxy carbonyl group attached to N-terminal end of hexapeptide demonstrated highest binding affinity amongst all the evaluated compounds. The compound identified in the study can be subjected further for in vitro and in vivo studies for ADMET properties.