Effect of High Concentration of Inert Cosolutes on the Refolding of an Enzyme

The kinetics of refolding of carbonic anhydrase II following transfer from a buffer containing 5 m guanidinium chloride to a buffer containing 0.5 m guanidinium chloride were studied by measuring the time-dependent recovery of enzymatic activity. Experiments were carried out in buffer containing concentrations of two “inert” cosolutes, sucrose and Ficoll 70, a sucrose polymer, at concentrations up to 150 g/liter. Data analysis indicates that both cosolutes significantly accelerate the rate of refolding to native or compact near-native conformations, but decrease the fraction of catalytically active enzyme recovered in the limit of long time. According to the simplest model that fits the data, both cosolutes accelerate a competing side reaction yielding inactive compact species. Acceleration of the side reaction by Ficoll is significantly greater than that of sucrose at equal w/v concentrations.

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The Influence of Varying Fluorination Patterns on the Thermodynamics and Kinetics of Benzenesulfonamide Binding to Human Carbonic Anhydrase II

Biomolecules ◽

10.3390/biom10040509 ◽

2020 ◽

Vol 10 (4) ◽

pp. 509 ◽

Cited By ~ 2

Author(s):

Steffen Glöckner ◽

Khang Ngo ◽

Björn Wagner ◽

Andreas Heine ◽

Gerhard Klebe

Keyword(s):

Carbonic Anhydrase ◽

Carbonic Anhydrase Ii ◽

The Novel ◽

Binding Modes ◽

X Ray ◽

Structure Activity ◽

Small Set ◽

Novel Method ◽

Human Carbonic Anhydrase ◽

Kinetics Of

The fluorination of lead-like compounds is a common tool in medicinal chemistry to alter molecular properties in various ways and with different goals. We herein present a detailed study of the binding of fluorinated benzenesulfonamides to human Carbonic Anhydrase II by complementing macromolecular X-ray crystallographic observations with thermodynamic and kinetic data collected with the novel method of kinITC. Our findings comprise so far unknown alternative binding modes in the crystalline state for some of the investigated compounds as well as complex thermodynamic and kinetic structure-activity relationships. They suggest that fluorination of the benzenesulfonamide core is especially advantageous in one position with respect to the kinetic signatures of binding and that a higher degree of fluorination does not necessarily provide for a higher affinity or more favorable kinetic binding profiles. Lastly, we propose a relationship between the kinetics of binding and ligand acidity based on a small set of compounds with similar substitution patterns.

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Kinetics of Enzymes with Iso-Mechanisms: Dead-End Inhibition of Fumarase and Carbonic Anhydrase II

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.1994.1303 ◽

1994 ◽

Vol 312 (1) ◽

pp. 227-233 ◽

Cited By ~ 16

Author(s):

K.L. Rebholz ◽

D.B. Northrop

Keyword(s):

Carbonic Anhydrase ◽

Carbonic Anhydrase Ii ◽

Dead End ◽

Kinetics Of

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The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

Biochemical Journal ◽

10.1042/bj20070708 ◽

2007 ◽

Vol 407 (3) ◽

pp. 397-406 ◽

Cited By ~ 23

Author(s):

Victoria J. Just ◽

Matthew R. Burrell ◽

Laura Bowater ◽

Iain McRobbie ◽

Clare E. M. Stevenson ◽

...

Keyword(s):

Crystal Structures ◽

Active Site ◽

Structural Information ◽

Side Reaction ◽

Oxalate Oxidase ◽

Decarboxylase Activity ◽

Oxalate Decarboxylase ◽

Catalytically Active ◽

The Stability ◽

Kinetics Of

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural.

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Carbonic anhydrase in human platelets

Biochemical Journal ◽

10.1042/bj2170727 ◽

1984 ◽

Vol 217 (3) ◽

pp. 727-730 ◽

Cited By ~ 14

Author(s):

W Siffert ◽

G Gros

Keyword(s):

Carbonic Anhydrase ◽

Carbonic Anhydrase Activity ◽

Human Platelets ◽

Carbonic Anhydrase Ii ◽

Affinity Column ◽

Kinetic Properties ◽

Km Value ◽

Flow Apparatus ◽

High Concentrations ◽

Kinetics Of

The carbonic anhydrase activity of human platelets was investigated by measuring the kinetics of CO2 hydration in supernatants of platelet lysates by using a pH stopped-flow apparatus. An average carbonic anhydrase concentration of 2.1 microM was determined for pellets of human platelets. Analysis of the kinetic properties of this carbonic anhydrase yielded a Km value of 1.0 mM, a catalytic-centre activity kcat. of 130000 s-1 and an inhibition constant Ki towards ethoxzolamide of 0.3 nM. From these values, CO2 hydration inside platelets is estimated to be accelerated by a factor of 2500. When platelet lysates were subjected to affinity chromatography, only the high-activity carbonic anhydrase II could be eluted from the affinity column, whereas the carbonic anhydrase isoenzyme I, which is known to occur in high concentrations in human erythrocytes, appeared to be absent.

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