Kinetic aspects of conformational changes in proteins. I. Rate of regain of enzyme activity from denatured proteins

Biochemistry ◽  
1971 ◽  
Vol 10 (5) ◽  
pp. 792-798 ◽  
Author(s):  
John W. Teipel ◽  
Daniel E. Koshland
Keyword(s):  
Enzyme Activity ◽  
Conformational Changes ◽  
Denatured Proteins

Activity and conformational changes of horseradish peroxidase in trifluoroethanol

2002 ◽  
Vol 80 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Hong-Wei Zhou ◽  
Yan Xu ◽  
Hai-Meng Zhou
Keyword(s):  
Enzyme Activity ◽  
Horseradish Peroxidase ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intrinsic Fluorescence ◽  
Two Phase ◽  
Main Effect ◽  
Α Helix ◽  
Kinetics Of

The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5–25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25–40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The α-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.Key words: horseradish peroxidase, trifluoroethanol, unfolding, Soret.

scholarly journals Crystal structure of pyrrolizidine alkaloid N-oxygenase from the grasshopper Zonocerus variegatus

2018 ◽  
Vol 74 (5) ◽  
pp. 422-432 ◽  
Author(s):  
Christian Kubitza ◽  
Annette Faust ◽  
Miriam Gutt ◽  
Luzia Gäth ◽  
Dietrich Ober ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Conformational Changes ◽  
Pyrrolizidine Alkaloid ◽  
Chemical Defence ◽  
Kinetic Studies ◽  
Food Plants ◽  
Single Amino Acid ◽  
Dimensional Structure ◽  
Zonocerus Variegatus

The high-resolution crystal structure of the flavin-dependent monooxygenase (FMO) from the African locust Zonocerus variegatus is presented and the kinetics of structure-based protein variants are discussed. Z. variegatus expresses three flavin-dependent monooxygenase (ZvFMO) isoforms which contribute to a counterstrategy against pyrrolizidine alkaloids (PAs). PAs are protoxic compounds produced by some angiosperm lineages as a chemical defence against herbivores. N-Oxygenation of PAs and the accumulation of PA N-oxides within their haemolymph result in two evolutionary advantages for these insects: (i) they circumvent the defence mechanism of their food plants and (ii) they can use PA N-oxides to protect themselves against predators, which cannot cope with the toxic PAs. Despite a high degree of sequence identity and a similar substrate spectrum, the three ZvFMO isoforms differ greatly in enzyme activity. Here, the crystal structure of the Z. variegatus PA N-oxygenase (ZvPNO), the most active ZvFMO isoform, is reported at 1.6 Å resolution together with kinetic studies of a second isoform, ZvFMOa. This is the first available crystal structure of an FMO from class B (of six different FMO subclasses, A–F) within the family of flavin-dependent monooxygenases that originates from a more highly developed organism than yeast. Despite the differences in sequence between family members, their overall structure is very similar. This indicates the need for high conservation of the three-dimensional structure for this type of reaction throughout all kingdoms of life. Nevertheless, this structure provides the closest relative to the human enzyme that is currently available for modelling studies. Of note, the crystal structure of ZvPNO reveals a unique dimeric arrangement as well as small conformational changes within the active site that have not been observed before. A newly observed kink within helix α8 close to the substrate-binding path might indicate a potential mechanism for product release. The data show that even single amino-acid exchanges in the substrate-entry path, rather than the binding site, have a significant impact on the specific enzyme activity of the isoforms.

scholarly journals Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison’s disease

2005 ◽  
Vol 152 (1) ◽  
pp. 95-101 ◽  
Author(s):  
L Nikfarjam ◽  
S Kominami ◽  
T Yamazaki ◽  
S Chen ◽  
R Hewer ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Enzyme Activity ◽  
Cytochrome P450 ◽  
Conformational Changes ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Cytochrome P450 Reductase ◽  
Difference Spectra ◽  
Native Form ◽  
Mechanism Of Inhibition

Objective: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. Design: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. Methods: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. Results: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450 nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. Conclusions: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

scholarly journals Structural insights into SETD3-mediated histidine methylation on β-actin

2018 ◽  
Author(s):  
Qiong Guo ◽  
Shanhui Liao ◽  
Sebastian Kwiatkowski ◽  
Weronika Tomaka ◽  
Huijuan Yu ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Enzyme Activity ◽  
Small Molecule ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Domain Protein ◽  
First Time ◽  
Structural Insights

SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Recently, we have identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin. Here we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses supported by the site-directed mutagenesis experiments and the enzyme activity assays indicated that the recognition and methylation of β-actin by SETD3 is highly sequence specific, and both SETD3 and β-actin adopt pronounce conformational changes upon binding to each other. In conclusion, the data show for the first time a catalytic mechanism of SETD3-mediated histidine methylation in β-actin, which not only throws light on protein histidine methylation phenomenon, but also facilitates the design of small molecule inhibitors of SETD3.

scholarly journals Contribution of Distinct Structural Elements to Activation of Calpain by Ca2+Ions

2004 ◽  
Vol 279 (19) ◽  
pp. 20118-20126 ◽  
Author(s):  
Anita Alexa ◽  
Zoltán Bozóky ◽  
Attila Farkas ◽  
Peter Tompa ◽  
Peter Friedrich
Keyword(s):  
Enzyme Activity ◽  
Conformational Changes ◽  
Site Directed Mutagenesis ◽  
Schematic Model ◽  
Structural Elements ◽  
Long Distance ◽  
Relay System ◽  
Domain Iii ◽  
A Site ◽  
Mutagenesis Study

The effect of Ca2+in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+binding and Ca2+sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+relay system. Wild-typeDrosophilaCalpain B domain III binds two to three Ca2+ions with aKdof 3400 μm. Phospholipids lower this value to 220 μm. Ca2+binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+need of both enzymes, lowering half-maximal Ca2+concentration from 8.6 to 1.3 mmfor Calpain B and from 250 to 7 μmfor m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this “extended transducer” mechanism is presented.

Inactivation and conformational changes of creatine kinase at low concentrations of hexafluoroisopropanol solutions

2003 ◽  
Vol 81 (5) ◽  
pp. 327-333 ◽  
Author(s):  
Xiao-Yun Wang ◽  
Fan-Guo Meng ◽  
Hai-Meng Zhou
Keyword(s):  
Creatine Kinase ◽  
Enzyme Activity ◽  
Conformational Changes ◽  
Fluorescence Spectra ◽  
Cd Spectra ◽  
Irreversible Inhibition ◽  
Low Concentrations ◽  
Flexible Region ◽  
Far Ultraviolet ◽  
Kinetics Of

Using the methods of far-ultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays, the inactivation and conformational changes of creatine kinase (CK) induced by 1,1,1,3,3,3-hexafluoro-2-propanol (hexafluoroisopropanol (HFIP)) of different concentrations were investigated. To avoid the aggregation of CK that occurs with high HFIP, concentrations of 0%–5% HFIP were used in this study. The CD spectra showed that HFIP concentrations above 2.5% strongly induced the formation of secondary structures of CK. No marked conformational changes were observed at low concentrations of HFIP (0%–2.5%). After incubation with 0.2% HFIP for 10 min, CK lost most of its activity. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou was applied to study the kinetics of CK inactivation during denaturation by HFIP. The inactivation rate constants for the free enzyme and the substrate–enzyme complex were determined by Tsou's method. The results suggested that low concentrations of HFIP had a high potential to induce helices of protein and that the active site of the enzyme was situated in a limited and flexible region of the enzyme molecule that was more susceptible to the denaturant than was the protein as a whole.Key words: creatine kinase, inactivation, conformation, kinetics, hexafluoroisopropanol.

Surface modification mediates the interaction between fullerene and lysozyme: protein structure and antibacterial activity

2021 ◽  
Author(s):  
Yitong Bai ◽  
Xian Wu ◽  
Peng Ouyang ◽  
Mengyao Shi ◽  
Qun Li ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Enzyme Activity ◽  
Surface Modification ◽  
Protein Structure ◽  
Conformational Changes ◽  
Hydrophilic Surface ◽  
Protein Conformational Changes ◽  
Activity Loss

Hydrophilic surface modification enhanced the interaction between fullerene and lysozyme to induce more protein conformational changes and enzyme activity loss.

scholarly journals Ligand-induced Conformational Changes within a Hexameric Acyl-CoA Thioesterase

2011 ◽  
Vol 286 (41) ◽  
pp. 35643-35649 ◽  
Author(s):  
Mary Marfori ◽  
Bostjan Kobe ◽  
Jade K. Forwood
Keyword(s):  
Enzyme Activity ◽  
Conformational Changes ◽  
Coenzyme A ◽  
Quaternary Structure ◽  
Mutational Analysis ◽  
Structural Data ◽  
Fatty Acyl ◽  
Bacillus Halodurans ◽  
Structural Level ◽  
Hot Dog

Acyl-coenzyme A (acyl-CoA) thioesterases play a crucial role in the metabolism of activated fatty acids, coenzyme A, and other metabolic precursor molecules including arachidonic acid and palmitic acid. These enzymes hydrolyze coenzyme A from acyl-CoA esters to mediate a range of cellular functions including β-oxidation, lipid biosynthesis, and signal transduction. Here, we present the crystal structure of a hexameric hot-dog domain-containing acyl-CoA thioesterase from Bacillus halodurans in the apo-form and provide structural and comparative analyses to the coenzyme A-bound form to identify key conformational changes induced upon ligand binding. We observed dramatic ligand-induced changes at both the hot-dog dimer and the trimer-of-dimer interfaces; the dimer interfaces in the apo-structure differ by over 20% and decrease to about half the size in the ligand-bound state. We also assessed the specificity of the enzyme against a range of fatty acyl-CoA substrates and have identified a preference for short-chain fatty acyl-CoAs. Coenzyme A was shown both to negatively regulate enzyme activity, representing a direct inhibitory feedback, and consistent with the structural data, to destabilize the quaternary structure of the enzyme. Coenzyme A-induced conformational changes in the C-terminal helices of enzyme were assessed through mutational analysis and shown to play a role in regulating enzyme activity. The conformational changes are likely to be conserved from bacteria through to humans and provide a greater understanding, particularly at a structural level, of thioesterase function and regulation.

scholarly journals Chemical modification of aminopeptidase isolated from Pronase

1994 ◽  
Vol 302 (2) ◽  
pp. 595-600 ◽  
Author(s):  
S H Yang ◽  
C H Wu ◽  
W Y Lin
Keyword(s):  
Enzyme Activity ◽  
Chemical Modification ◽  
Metal Ions ◽  
Conformational Changes ◽  
Enzyme Catalysis ◽  
Competitive Inhibitor ◽  
Amino Groups ◽  
Active Centre ◽  
Modified Enzyme

Chemical modification of aminopeptidase from pronase has revealed two important histidines in enzyme catalysis. In the absence of metal ions, modification of the readily-modified histidine (pKa 6.9 +/- 0.5) results in a drastic loss of activity, indicating that this residue is indispensible for enzyme activity. In the presence of CaCl2, the modified enzyme still retains approx. 60% of the activity, whereas modification of another histidine (pKa 7.7 +/- 0.2) leads to a dramatic loss of activity. In fact, the enzyme with the first histidine being modified is active only in the presence of metal ions. Moreover, modification of the second histidine is prevented by the presence of Ca(II). These results indicate that the second histidine is serving as a ligand for Ca(II) and the bound Ca(II) is directly involved in enzyme catalysis. The c.d. spectra of the modified and unmodified enzymes in the absence or presence of CaCl2 are all very similar, indicating that no gross conformational changes in protein occur upon modification or by the presence of Ca(II). Modification of both histidines is prevented by the presence of a competitive inhibitor, suggesting that they are located in the active centre. Modification of 11 amino groups, two tyrosines, or four arginines causes no appreciable inactivation of the enzyme, indicating that these residues are not directly involved in enzyme catalysis.

Sign in / Sign up

Export Citation Format

Share Document