• BMB Reports
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• v.31 no.4
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• pp.307-327
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• 1998

Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme which catalyzes the reduction of hydrogen peroxide to water using two equivalents of ferrocytochrome c. The CcP/cytochrome c system has many features which make it a very useful model for detailed investigation of heme protein structure/function relationships including activation of hydrogen peroxide, protein-protein interactions, and long-range electron transfer. Both CcP and cytochrome c are single heme, single subunit proteins of modest size. High-resolution crystallographic structures of both proteins, of one-to-one complexes of the two proteins, and a number of active-site mutants are available. Site-directed mutagenesis studies indicate that the distal histidine in CcP is primarily responsible for rapid utilization of hydrogen peroxide implying significantly different properties of the distal histidine in the peroxidases compared to the globins. CcP and cytochrome c bind to form a dynamic one-to-one complex. The binding is largely electrostatic in nature with a small, unfavorable enthalpy of binding and a large positive entropy change upon complex formation. The cytochrome c-binding site on CcP has been mapped in solution by measuring the binding affinities between cytochrome c and a number of CcP surface mutations. The binding site for cytochrome c in solution is consistent with the crystallographic structure of the one-to-one complex. Evidence for the involvement of a second, low-affinity cytochrome c-binding site on CcP in long-range electron transfer between the two proteins is reviewed.

• Archives of Pharmacal Research
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• v.14 no.3
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• pp.231-236
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• 1991

The high affinity binding sites for angiotensin II were solubilized from rat liver membranes by treatment with CHAPS. The binding protein was also partially purified by angiotensin III inhibitor-coupled Affi-gel affinity chromatography. Binding to the intact membrances as well as to the solubilized preparation was specific and saturable. According to the Scatchard plot, the membrane preparations exhibited a single class of high affinity binding sites with a Kd OF 0.71 nM. The solubilized preparation also showed the presence of a single class of bindings sites with less affinity (Kd of 14 nM). Meanwhile the competition studies using angiotensin II analogues represented two separate binding sites for angiotensin II and single binding site for antagonist. These latter findings were correlated to the results provided by Garrison's research group. More works are needed to clarify this discrepancy.

• BMB Reports
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• v.28 no.4
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• pp.290-292
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• 1995

Existence of a binding site for choline esters with an acyl chain of various sizes was examined by comparing the inhibitory potency of the respective compound. In contrast to acetylcholine, which showed a pure competitive pattern of inhibition, choline esters with an acyl chain of a long size ($C{\geq}5$) expressed a mixed type of inhibition. Binding of choline esters containing a long chain ($C_7-C_{12}$) to the hydrophobic region in the active site is deduced from a linear relationship between the $K_{iE}$ value and the size of acyl moiety, and a good hydrophobicity relationship. In addition, the non-competitive component in the inhibition of acetylcholinesterase seems to be due to the interaction of choline esters with both the hydrophobic site and the trimethylammonium-binding site in the active center of the acetylated acetylcholinesterase.

• Bulletin of the Korean Chemical Society
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• v.21 no.8
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• pp.779-784
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• 2000

Comparing the adsorption properties and dissociation on a Pt(111) iththat ona Fe(111) surface, we have con-sidered seven coordination modes of the adsorbed binding site: $di-${\sigma}$${\Delta}$\mu\pi/\mu$, 1-fbld,2-fold, and 3-fbld sites. On the Pt(111) surface, t he adsorbed binding site of carbon dioxide was strongestat the1-fold site and weakest at the $\pi/\mu-site.$ The adsorbed binding site on the Fe(111) surface was strongest at the di-бsite and weakest at the 3-fold site. We have found that the binding energy at each site that excepted 3-fold on the Fe(111) surface was stronger than the binding energy on the Pt(111) surface and that chemisorbed $CO_2bends$ because of metal mixing with $2\piu${\rightarrow}$6a_1CO_2orbital.$, The dissociation reaction occured in two steps, with an intermediate com-plex composed of atomic oxygen and ${\pi}bonding$ CO forming. The OCO angles of reaction intermediate com-plex structure for the dissociation reaction $were115^{\circ}Con$ the Pt(111), and $117^{\circ}C$ on the Fe(111) surface. We have found that the $CO_2dissociation$ rea11) surface proceeds easily,with an activationenergy about 0.2 eV lower than that on the Pt(111) surface.

• BMB Reports
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• v.28 no.2
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• pp.94-99
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• 1995

The properties of binding sites in the active site of $Zn^{2+}$-glycerophosphocholine cholinephosphodiesterase were examined using substrates and inhibitors of the enzyme. Phosphodiesterase hydrolyzed p-nitrophenylphosphocholine, p-aminophenylphosphocholine, and glycerophosphocholine, but did not hydrolyze either acylated glycerophosphocholine or bis (p-nitrophenyl)phosphate, suggesting a size limitation for interaction with a glyceryl moiety-binding subsite. The hydrolysis of p-nitrophenylphosphocholine was competitively inhibited by glycerophosphocholine and p-aminophenylphosphocholine, while glycerophosphoethanolamine was a weak inhibitor. The enzyme was also inhibited by choline, but not by ethanolamine. Thiocholine, a much more potent inhibitor than choline, was more inhibitory than cysteamine, suggesting a strict specificity of an anionic subsite adjacent to a $Zn^{2+}$ subsite. Of all oxyanions tested, the tellurite ion was found to strongly inhibit the enzyme by binding to a $Zn^{2+}$ subsite. The inhibitory role of tellurite was synergistically enhanced by tetraalkylammonium salts, but not by glycerol. Deactivation of the enzyme by diethylpyrocarbonate was partially protected by choline, but not by glycerophosphate. It is suggested that the active site of phosphodiesterase contains three binding subsites.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.10a
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• pp.9-15
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• 1995

When quiescent cells ate stimulated to enter the cell cycle, the thymidine kinase(TK) gene is transcriptionally activated at the border of Gl and 5. In this report we show that the human TK promoter contains multiple protein-binding sites. By site-directed mutagenesis, we identified a protein-binding site on the human TK promoter requited for conferring Gl-S-regulated transcription to a heterologous promoter and dissociated it functionally from an adjacent protein-binding domain containing an inverted CCAAT motif requited for high basal level expression. Substitution-mutation of this site results in constitutive expression of the neo reporter gene in serum-stimulated fibroblasts, as well as in cells arrested in mid-Gl by a temperature-sensitive mutation. The regulatory domains for the human TK promoter exhibit interesting symmetrical features, including a set of CCAAT motifs and sites similar to the novel Yi protein-binding site recently discovered in the mouse TK promoter. Thus, components of the hTK complex is important for hTK gene regulation.

• YAKHAK HOEJI
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• v.28 no.3
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• pp.129-138
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• 1984

Specific [$^{3}H$] ouabain binding and $Ca^{2+}$ -uptake were measured to elucidate the role of high affinity [$^{3}H$] ouabain binding site in rat cardiac myocytes which contain 65% of rod cells. High affinity [$^{3}$H] ouabain binding site, which is about 3% of total pump sites, with apparent dissociation constant ($K_{D}$) of $1.1{\times}10^{-7}M$ and maximum binding site concentration (Bmax) of 1.2 pmol/mg protein ($1.754{\times}10^{5}cells$) were identified. At the concentration of $10^{-7}M$ to $10^{-4}M$, ouabain produced concentration dependent increase in $Ca^{2+}$-uptake of myocytes. The effect of ouabain on $Ca^{2+}$-uptake was not effected by membrane depolarization (elevated K+ in incubation medium) or verapamil. These results suggest that in rat ventricular myocytes the ouabain receptor complex to high affinity site may increase Na+ - $Ca^{2+}$ exchange across the sarcolemmal membrane by inhibition of Na+, K+ - ATPase.

• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2248-2252
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• 2007

SOMT-9 is an O-methyltransferase that utilizes quercetin to produce 3'-methoxy quercetin. In order to determine which amino acids of SOMT-9 are important for this reaction and its regioselectivity, molecular docking experiments followed by site directed mutagenesis were performed. Molecular modeling and molecular docking experiments identified several amino acid residues involved in metal binding, AdoMet binding, and substrate binding. Site-directed mutagenesis showed that Asp188 is critical for metal binding and that Lys165 assists other metal binding residues in maintaining quercetin in the proper position during the reaction. In addition, Tyr207 was shown to play an important role in the determination of the regioselectivity and Met60 was shown to be involved in formation of the hydrophobic pocket necessary for substrate binding. The molecular modeling and docking experiments discussed in this study could be applicable to future research including prediction of substrate binding and regioselectivity of an enzyme.

• BMB Reports
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• v.30 no.5
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• pp.315-319
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• 1997

It was discovered that monoclonal anti-erythropoietin (EPO) antibody CFC-6 can neutralize EPO-induced cell activation. To know the binding site of CFC-6, recombinant human erythropoietin (rhEPO) was digested with Glu-C, followed by a separation using high performance liquid chromato graphy (HPLC). Each HPLC fraction was blotted on the nitrocellulose membrane and the membrane was treated with anti-EPO antibody CFC-6 and anti-mouse antibody which is modified with peroxidase. Only one spot showed the color and the fraction was sequenced by Edman degradation. The results suggest that CFC-6 recognizes amino acid sequence V63-W-Q-G-L-A-L-L-S-E72 which is a part of helix II of the EPO molecule. Binding of CFC-6 to EPO may inhibit EPO binding to its receptor, which implies that the antibody binding site and the receptor binding site are close or overlapping.

• Proceedings of the KIEE Conference
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• 1987.07a
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• pp.493-496
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• 1987

Theoretical analysis was performed to understand the sensing mechanism of pH-ISFET by using the site binding model. The calculated pH-${\varphi}$ relationship showed good fittings with the experimental results.