Chemical reactivity of the active site of myoglobin

1. Chloroacetone (I) was shown to be an active-site-directed inhibitor of the aliphatic amidase (EC 3.5.1.4) from Pseudomonas aeruginosa strain PAC142.2. This inhibitor reacted with the enzyme in two stages: the first involving the reversible formation of an enzymically inactive species, EI, and the second the formation of a species, EX, from which enzymic activity could not be recovered. 3. Different types of kinetic experiment were conducted to test conformity of the reaction to the scheme: E + I k+1 Equilibrium k-1 EI Leads to K+2 EX A computer-based analysis of the results was carried out and values of the individual rate constants were determined. 4. No direct evidence for a binding step before the formation of EI could be obtained, as with [E]0 Less Than [I]0 the observed first-order rate constant for the formation of EI was directly proportional to the concentration of chloroacetone up to 1.2 mM (above this concentration the reaction became too rapid to follow even by the stopped-flow method developed to investigate fast inhibition). 5. The value of k+1 exhibited a bell-shaped pH-dependency with a maximum value of about 3 × 10(3) M-1. S-1 at pH6 and apparent pKa values of 7.8 and about 4.8.6. The values of k-1 and K+2 were similar and changed with the time of reaction from values of about 3 × 10(-3) S-1 (pH8.6) at short times to about one-sixth this value for longer periods of incubation. In this respect the simple reaction scheme is insufficient to describe the inhibition process. 7. The overall inhibition reaction is rapid, whether it is considered in relation to the expected chemical reactivity of chloroacetone, the rate of reaction of other enzymes with substrate analogues containing the chloromethyl group, or the rate of the amidase-catalysed hydrolysis of N-methylacetamide, a substrate that is nearly isosteric with chloroacetone. 8. Acetamide protected the amidase from inhibition by chloroacetone, and the concentration-dependence of the protection gave a value of an apparent dissociation constant similar to the Km value for this substrate. 9. Addition of acetamide to solutions of the species EI led to a slow recovery of activity. Recovery of active enzyme was also observed after dilution of a solution of EI in the absence of substrate. 10. The species EI is considered not to be a simple adsorption complex, and the possibilities are discussed that it may be a tetrahedral carbonyl adduct, a Schiff base (azomethine) or a complex in which the enzyme has undergone a structural change. The species EX is probably a derivative in which there is a covalent bond between a group in the enzyme and the C-1 atom of the inhibitor.

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Engineering the active site of ascorbate peroxidase

Biochemical Society Transactions ◽

10.1042/bst0290105 ◽

2001 ◽

Vol 29 (2) ◽

pp. 105-111 ◽

Cited By ~ 3

Author(s):

E. Lloyd Raven ◽

A. Celik ◽

P. M. Cullis ◽

R. Sangar ◽

M. J. Sutcliffe

Keyword(s):

Cytochrome P450 ◽

Protein Structure ◽

Ascorbate Peroxidase ◽

Active Site ◽

Chemical Reactivity ◽

General Context ◽

Catalytic Chemistry

Understanding the catalytic versatility of haem enzymes, and in particular the relationships that exist between different classes of haem-containing proteins and the mechanisms by which the apo-protein structure controls chemical reactivity, presents a major experimental and theoretical challenge. These issues are discussed in the general context of peroxidase and cytochrome P450 chemistry, and specific issues relating to the catalytic chemistry of ascorbate peroxidase are highlighted.

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A convenient synthesis of labelled rhodopsin and studies on its active site

Biochemical Journal ◽

10.1042/bj1190359 ◽

1970 ◽

Vol 119 (3) ◽

pp. 359-366 ◽

Cited By ~ 18

Author(s):

M. D. Hirtenstein ◽

M. Akhtar

Keyword(s):

Conformational Change ◽

Sodium Borohydride ◽

Active Site ◽

Cetyltrimethylammonium Bromide ◽

Chemical Reactivity ◽

Amino Group ◽

Polar Environment ◽

Convenient Synthesis ◽

First Time ◽

Native Site

Digitonin solutions of labelled rhodopsin, containing 3H in the retinyl moiety, were prepared by two related methods. Labelled rhodopsin was also prepared for the first time in cetyltrimethylammonium bromide and purified by column chromatography. It was shown that only certain rhodopsin preparations on denaturation in the dark and the reduction with sodium borohydride gave up to 60% of the radioactivity in a fraction characterized as N-retinylphosphatidylethanolamine. Such preparations also gave a lipid-linked retinyl moiety at the metarhodopsin-I stage, but, as expected, a protein-linked retinyl moiety at the metarhodopsin-II stage. Other preparations however, gave exclusively protein-bound radioactivity at the native-rhodopsin, metarhodopsin-I and metarhodopsin-II stages. It is therefore conceivable that the formation of N-retinylphosphatidylethanolamine is due to a non-enzymic reaction resulting from the transfer of the retinyl moiety from its native site to an amino group of a favourably oriented phospholipid molecule. The only firmly established aspect of the rhodopsin active site remains the demonstration in our previous work that at the metarhodopsin-II stage the retinyl moiety is linked to an ∈-amino group of lysine. On the basis of chemical reactivity it is argued that the light-induced conversion of rhodopsin into metarhodopsin II involves a profound conformational change resulting in the dislocation of the retinylideneiminium chromophore from a non-polar environment in rhodopsin to a polar environment in metarhodopsin II.

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The Oxidative Process of Acarbose, Maysin, and Luteolin with Maltase-Glucoamylase: Molecular Docking and Molecular Dynamics Study

Applied Sciences ◽

10.3390/app11094067 ◽

2021 ◽

Vol 11 (9) ◽

pp. 4067

Author(s):

Linda-Lucila Landeros-Martínez ◽

Néstor Gutiérrez-Méndez ◽

Juan Pedro Palomares-Báez ◽

Nora-Aydeé Sánchez-Bojorge ◽

Juan Pablo Flores-De los Ríos ◽

...

Keyword(s):

Molecular Dynamics ◽

Amino Acids ◽

Molecular Docking ◽

Active Site ◽

Density Functional ◽

Chemical Reactivity ◽

Basis Set ◽

Electronic Charge ◽

Chemical Hardness ◽

Oxidative Process

Type 2 diabetes mellitus has been classified as the epidemic of the XXI century, making it a global health challenge. Currently, the commonly used treatment for this disease is acarbose, however, the high cost of this medicine has motivated the search for new alternatives. In this work, the maysin, a characteristic flavonoid from maize inflorescences, and its aglycon version, luteolin, are proposed as acarbose substitutes. For this, a theoretical comparative analysis was conducted on the molecular interactions of acarbose, maysin, and luteolin with human maltase-glucoamylase (NtMGAM), as well as their oxidative process. The binding energies in the active site of NtMGAM with acarbose, maysin, and luteolin molecules were predicted using a molecular docking approach applying the Lamarckian genetic algorithm method. Theoretical chemical reactivity parameters such as chemical hardness (η) and chemical potential (µ) of the acarbose, maysin, and luteolin molecules, as well as of the amino acids involved in the active site, were calculated using the electronic structure method called Density Functional Theory (DFT), employing the M06 meta-GGA functional in combination with the 6-31G(d) basis set. Furthermore, a possible oxidative process has been proposed from quantum-chemical calculations of the electronic charge transfer values (ΔN), between the amino acids of the active site and the acarbose, maysin, and luteolin. Molecular docking predictions were complemented with molecular dynamics simulations. Hence, it was demonstrated that the solvation of the protein affects the affinity order between NtMGAM and ligands.

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Analysis of the Molecular Interactions between Cytochromes P450 3A4 and 1A2 and Aflatoxins: A Docking Study

Applied Sciences ◽

10.3390/app9122467 ◽

2019 ◽

Vol 9 (12) ◽

pp. 2467 ◽

Cited By ~ 2

Author(s):

Isui Abril García-Montoya ◽

Norma Rosario Flores-Holguín ◽

Linda-Lucila Landeros-Martínez ◽

Mónica Alvarado-González ◽

Quintín Rascón-Cruz ◽

...

Keyword(s):

Molecular Structure ◽

Amino Acids ◽

Molecular Docking ◽

Charge Transfer ◽

Active Site ◽

Density Functional ◽

Chemical Reactivity ◽

Cytochromes P450 ◽

Docking Analysis ◽

Molecular Docking Analysis

Mycotoxins known as aflatoxins (AF) are produced as a secondary metabolite by some species of Aspergillus fungi. They are considered carcinogenic, hepatotoxic, teratogenic, and mutagenic. In this study, the molecular structure, chemical reactivity, and charge transfer values of AFB1, B2, G1, and G2 were analyzed using density functional theory. Different methodologies—B3LYP/6-311G(d,p) and M06-2X/6-311G(d,p)—were applied for geometrical calculations. Chemical reactivity parameters were used in the calculation of charge transfer values during the interaction between protein and ligand. The binding energy, the electrostatic interactions, and the amino acids of the active site were determined by molecular docking analysis between AF and cytochromes P450 (3A4 and 1A2), employing different PDB files (CYP3A4:1TQN, 2V0M, 4NY4 and 1W0E, and CYP1A2:2HI4). Molecular docking analysis indicated that the central rings of the AF are involved in the interaction with the HEM group of the active site. The differences in the molecular structure of the AF affect their position regarding the HEM group. The resulting configurations presented considerable variation in the amino acids and the position of the coupling. The charge transfer values showed that there is oxidative damage inside the active site and that the HEM group is responsible for the main charge transferences.

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Angiotensin as a model for hormone – receptor interactions

Bioscience Reports ◽

10.1007/bf01116558 ◽

1985 ◽

Vol 5 (5) ◽

pp. 407-416 ◽

Cited By ~ 22

Author(s):

Graham J. Moore ◽

John M. Matsoukas

Keyword(s):

Magnetic Resonance ◽

Protease Inhibitors ◽

Active Site ◽

Serine Proteases ◽

Proton Magnetic Resonance ◽

Chemical Reactivity ◽

Receptor Activation ◽

Electronic Effects ◽

Relay System ◽

Receptor Interactions

Proton magnetic resonance and chemical reactivity studies have demonstrated the presence of a tyrosine charge relay system in angiotensin which is analogous to the serine charge relay system present at the active site of serine proteases. Receptor activation by angiotensin can be explained by electronic effects deriving from an interaction of the charge relay system with stacking of the histidine and phenylalanine rings. Experiments with serine protease inhibitors suggest the possibility that mechanistic features of the interaction of angiotensin with its receptors may apply to other ‘phenoxyl’ hormones including certain peptides, steroids and catecholamines.

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Spatially Resolved Photoelectron Spectroscopy: Recent Progress and Future Plans

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010013554x ◽

1990 ◽

Vol 48 (2) ◽

pp. 388-389

Author(s):

A. M. Bradshaw

Keyword(s):

Photoelectron Spectroscopy ◽

Chemical Reactivity ◽

Target Material ◽

Orbital Energy ◽

Light Sources ◽

Analytical Technique ◽

Hartree Fock ◽

Spatially Resolved ◽

Koopmans Theorem ◽

Surface Analytical Technique

X-ray photoelectron spectroscopy (XPS or ESCA) was not developed by Siegbahn and co-workers as a surface analytical technique, but rather as a general probe of electronic structure and chemical reactivity. The method is based on the phenomenon of photoionisation: The absorption of monochromatic radiation in the target material (free atoms, molecules, solids or liquids) causes electrons to be injected into the vacuum continuum. Pseudo-monochromatic laboratory light sources (e.g. AlKα) have mostly been used hitherto for this excitation; in recent years synchrotron radiation has become increasingly important. A kinetic energy analysis of the so-called photoelectrons gives rise to a spectrum which consists of a series of lines corresponding to each discrete core and valence level of the system. The measured binding energy, EB, given by EB = hv−EK, where EK is the kineticenergy relative to the vacuum level, may be equated with the orbital energy derived from a Hartree-Fock SCF calculation of the system under consideration (Koopmans theorem).

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Cluster analysis for large data sets: applications to individual aerosol particles from the mid-pacific

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100132078 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1488-1489

Author(s):

Thomas W. Shattuck ◽

James R. Anderson ◽

Neil W. Tindale ◽

Peter R. Buseck

Keyword(s):

Cluster Analysis ◽

Chemical Reactivity ◽

Large Data ◽

Large Data Sets ◽

Particle Analysis ◽

Data Sets ◽

Halogen Chemistry ◽

Complete Study ◽

Components Analysis ◽

Automated Scanning

Individual particle analysis involves the study of tens of thousands of particles using automated scanning electron microscopy and elemental analysis by energy-dispersive, x-ray emission spectroscopy (EDS). EDS produces large data sets that must be analyzed using multi-variate statistical techniques. A complete study uses cluster analysis, discriminant analysis, and factor or principal components analysis (PCA). The three techniques are used in the study of particles sampled during the FeLine cruise to the mid-Pacific ocean in the summer of 1990. The mid-Pacific aerosol provides information on long range particle transport, iron deposition, sea salt ageing, and halogen chemistry.Aerosol particle data sets suffer from a number of difficulties for pattern recognition using cluster analysis. There is a great disparity in the number of observations per cluster and the range of the variables in each cluster. The variables are not normally distributed, they are subject to considerable experimental error, and many values are zero, because of finite detection limits. Many of the clusters show considerable overlap, because of natural variability, agglomeration, and chemical reactivity.

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Locating Al in the DABCO catalyst before and after Al extraction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100174461 ◽

1990 ◽

Vol 48 (4) ◽

pp. 265-267

Author(s):

Kathleen B. Reuter

Keyword(s):

Active Site ◽

Inverse Relationship ◽

Transverse Direction ◽

Reaction Rate ◽

Acid Phosphate ◽

Fracture Surfaces ◽

Al Content ◽

Before And After ◽

Relationship Of

The reaction rate and efficiency of piperazine to 1,4-diazabicyclo-octane (DABCO) depends on the Si/Al ratio of the MFI topology catalysts. The Al was shown to be the active site, however, in the Si/Al range of 30-200 the reaction rate increases as the Si/Al ratio increases. The objective of this work was to determine the location and concentration of Al to explain this inverse relationship of Al content with reaction rate.Two silicalite catalysts in the form of 1/16 inch SiO2/Al2O3 bonded extrudates were examined: catalyst A with a Si/Al of 83; and catalyst B, the acid/phosphate Al extracted form of catalyst A, with a Si/Al of 175. Five extrudates from each catalyst were fractured in the transverse direction and particles were obtained from the fracture surfaces near the center of the extrudate diameter. Particles were also obtained from the outside surfaces of five extrudates.

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Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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