scholarly journals Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence

2004 ◽  
Vol 380 (3) ◽  
pp. 627-633 ◽  
Author(s):  
Daniel ROCHU ◽  
Nathalie VIGUIÉ ◽  
Frédérique RENAULT ◽  
David CROUZIER ◽  
Marie-Thérèse FROMENT ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Metal Cation ◽  
Active Site ◽  
Ph Dependence ◽  
Conformational Stability ◽  
Maximum Activity ◽  
Active Centre ◽  
Optimum Activity ◽  
Maximum Stability ◽  
Activation Phase

Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T≤35 °C, followed by an activation phase near 45–48 °C and then inactivation which was completed at 60 °C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different Tm values of 60.5–67 °C, 58–64 °C and 53–64 °C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.

scholarly journals Cathepsin B, the lysosomal thiol proteinase of calf liver

1969 ◽  
Vol 114 (4) ◽  
pp. 673-678 ◽  
Author(s):  
O. Snellman
Keyword(s):  
Cathepsin B ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Chelating Agent ◽  
Thiol Group ◽  
Maximum Activity ◽  
Active Centre ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Thiol Proteinase

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.

scholarly journals Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes

1976 ◽  
Vol 153 (2) ◽  
pp. 397-402 ◽  
Author(s):  
A Koj ◽  
J Chudzik ◽  
A Dubin
Keyword(s):  
Methyl Ester ◽  
Substrate Specificity ◽  
Rose Bengal ◽  
Ph Dependence ◽  
Maximum Activity ◽  
Enzyme Molecule ◽  
Active Centre ◽  
Pancreatic Elastase ◽  
Horse Blood ◽  
Nitrophenyl Ester

Two proteinases (2A and 2B) purified from the granular fraction of horse blood leucocytes degrade casein (Km values 12.8 and 6mg/ml respectively) with maximum activity at pH 7.4 and in the presence of 2m-urea. Urea-denatured haemoglobin, fibrinogen, albumin and resorcin/fuchsin-stained elastin are digested at a slower rate. The enzymes hydrolyse synthetic substrates of elastase, N-benzyloxycarbonyl-L-alanine 4-nitrophenyl ester (Km 0.114 and 0.178 mM) and N-acetyl-tri-L-alanine methyl ester (Km 5.55 and 0.98 mM), but they do not hydrolyse synthetic substrates of trypsin, chymotrypsin and thrombin. The examined proteinases are completely inhibited by 2 mM-di-isopropyl phosphorfluoridate and show a sensitivity to butyl and octyl isocyanates similar to that of pancreatic elastase. The pH-dependence of their photoinactivation in the presence of Rose Bengal indicates the presence of histidine in the active centre. Proteinase 2A rather insensitive to iodination by IC1 as is pancreatic elastase, whereas proteinase 2B is totally inactivated after incorporation of five iodine atoms per enzyme molecule.

scholarly journals The pH-dependence of the binding of competitive inhibitors to pepsin

1969 ◽  
Vol 113 (2) ◽  
pp. 343-351 ◽  
Author(s):  
J. R. Knowles ◽  
Hilary Sharp ◽  
P. Greenwell
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Equilibrium Dialysis ◽  
Ph Dependence ◽  
Binding Specificity ◽  
Electrostatic Repulsion ◽  
Conformation Change ◽  
Kinetic Measurements ◽  
Competitive Inhibitors ◽  
Ph Range

1. The pH-dependence of the binding to pepsin of four dipeptide competitive inhibitors is reported. Values of Ki obtained from equilibrium-dialysis experiments agree closely with those from kinetic measurements. 2. The binding of uncharged N-acyl-dipeptide amides to pepsin is essentially independent of pH from 0·2 to 5·8. Values of Ki for the corresponding N-acyl-dipeptide acids rise rapidly above pH3·5, and depend on the ionization of a group of apparent pKa 3·6. 3. The data indicate that pepsin does not undergo any gross conformation change (at least none that affects binding) over the whole pH range of its catalytic activity. The pH-dependence of the dipeptide acid inhibitors indicates that the acid anions do not bind to pepsin, presumably because of electrostatic repulsion between the inhibitor anion and a negative centre at or near the active site of the enzyme. 4. The binding of all four stereoisomers of N-acetylphenylalanylphenylalanine, of the depside analogues of the l–l- and d–l-compounds and of N-acetylglycyl-l-phenylalanine and N-acetyl-l-phenylalanylglycine was studied at pH2·2. 5. These results throw further light on the binding specificity of pepsin and on the charge nature of the active site of this enzyme.

scholarly journals Flower-Shaped C-Dots/Co3O4{111} Constructed with Dual-Reaction Centers for Enhancement of Fenton-Like Reaction Activity and Peroxymonosulfate Conversion to Sulfate Radical

Catalysts ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 135
Author(s):  
Zhibin Wen ◽  
Qianqian Zhu ◽  
Jiali Zhou ◽  
Shudi Zhao ◽  
Jinnan Wang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
High Surface ◽  
High Surface Area ◽  
Reaction Centers ◽  
Organic Radicals ◽  
The Other ◽  
High Catalytic Activity ◽  
High Adsorption ◽  
High Turnover

Novel flower-shaped C-dots/Co3O4{111} with dual-reaction centers were constructed to improve the Fenton-like reaction activity and peroxymonosulfate (PMS) conversion to sulfate radicals. Due to the exposure of a high surface area and Co3O4{111} facets, flower-shaped C-dots/Co3O4{111} could provide more Co(II) for PMS activation than traditional spherical Co3O4{110}. Meanwhile, PMS was preferred for adsorption on Co3O4{111} facets because of a high adsorption energy and thereby facilitated the electron transfer from Co(II) to PMS. More importantly, the Co–O–C linkage between C-dots and Co3O4{111} induced the formation of the dual-reaction center, which promoted the production of reactive organic radicals (R•). PMS could be directly reduced to SO4−• by R• over C-dots. On the other hand, electron transferred from R• to Co via Co–O–C linkage could accelerate the redox of Co(II)/(III), avoiding the invalid decomposition of PMS. Thus, C-dots doped on Co3O4{111} improved the PMS conversion rate to SO4−• over the single active site, resulting in high turnover numbers (TONs). In addition, TPR analysis indicated that the optimal content of C-dots doped on Co3O4{111} is 2.5%. More than 99% of antibiotics and dyes were degraded over C-dots/Co3O4{111} within 10 min. Even after six cycles, C-dots/Co3O4{111} still remained a high catalytic activity.

scholarly journals Probing the ionization state of substrate α-d-glucopyranosyl phosphate bound to glycogen phosphorylase b

1995 ◽  
Vol 308 (3) ◽  
pp. 1017-1023 ◽  
Author(s):  
I P Street ◽  
S G Withers
Keyword(s):  
Active Site ◽  
Glycogen Phosphorylase ◽  
Ph Dependence ◽  
Substrate Inhibition ◽  
19F Nmr ◽  
Ionization State ◽  
Nmr Studies ◽  
Substrate Analogues ◽  
Glycogen Phosphorylase B ◽  
Pka Value

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

Dehydration of n-Butanol on Phosphate-Modified Carbon Nanotubes: Active Site and Intrinsic Catalytic Activity

2021 ◽  
Author(s):  
Fan Li ◽  
Xueya Dai ◽  
Xingyu Lu ◽  
Chao Wang ◽  
Wei Qi
Keyword(s):  
Carbon Nanotubes ◽  
Catalytic Activity ◽  
Active Site ◽  
Efficient Utilization ◽  
Reaction Route ◽  
Modified Carbon Nanotubes

Dehydration of n-butanol (nB) to corresponding olefins (butene) is an important reaction route to realize the efficient utilization of bulk bio-alcohols. In this work, a novel phosphate modified oxidized multi-walled...

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

1986 ◽  
Vol 64 (4) ◽  
pp. 323-327 ◽  
Author(s):  
Mohammed A. Kashem ◽  
H. Brian Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Active Site ◽  
Nicotinamide Adenine Dinucleotide ◽  
Ph Dependence ◽  
Transient State ◽  
Nicotinamide Adenine ◽  
Compound I ◽  
Single Ionization ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Kinetics Of

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 °C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I–NADH reaction can be explained in terms of a single ionization of pKa = 4.7 ± 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-H–NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 ± 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II–NADH reaction was observed. Over the pH range of 4–10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 × 105 to5.6 × 102 M−1 s−1 and of HRP-II with NADH varied from 4.4 × 104 to 4.1 M−1 s−1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.

scholarly journals The interplay of electrostatic and binding interactions determining active centre chemistry and catalytic activity in actinidin and papain

1989 ◽  
Vol 257 (1) ◽  
pp. 309-310 ◽  
Author(s):  
K Brocklehurst ◽  
M O'Driscoll ◽  
D Kowlessur ◽  
I R Phillips ◽  
W Templeton ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Centre ◽  
Binding Interactions

scholarly journals Site-selective Protonation of the One-electron Reduced Cofactor in [FeFe]-Hydrogenase

2020 ◽  
Author(s):  
Konstantin Laun ◽  
Iuliia Baranova ◽  
Jifu Duan ◽  
Leonie Kertess ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
H2 Evolution ◽  
Proton Reduction ◽  
Ph Values ◽  
Site Selective ◽  
H2 Oxidation ◽  
Diiron Site

Hydrogenases are microbial redox enzymes that catalyze H2 oxidation and proton reduction (H2 evolution). While all hydrogenases show high oxidation activities, the majority of [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands that facilitate catalysis at low overpotential. Distinct proton transfer pathways connect the active site niche with the solvent, resulting in a non-trivial dependence of hydrogen turnover and bulk pH. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ in situ infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pKa differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the natural pH dependence of hydrogen turnover as catalyzed by [FeFe]-hydrogenase.<br>

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