Influence of enzyme conformational changes on catalytic activity investigated by circular dichroism spectroscopy

Rigos, Carolina Fortes; Santos, Hérica de Lima; Thedei, Geraldo; Ward, Richard J.; Ciancaglini, Pietro

doi:10.1002/bmb.2003.494031050264

Cited by 19 publications

(7 citation statements)

References 15 publications

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“…Changes in the secondary structure of ALP and LDH after HPP were studied using the far-ultraviolet CD spectrum in the 190–250 nm range. In general, protein activation is observed as a shift downward of the CD spectrum – , whereas inactivation shifts the CD spectrum upward – . A denatured protein is thus observed as an upward shift in ellipticity.…”

Section: Resultsmentioning

confidence: 99%

Effect of High-Pressure Processing on Activity and Structure of Alkaline Phosphatase and Lactate Dehydrogenase in Buffer and Milk

Kouassi

Anantheswaran

Knabel

et al. 2007

J. Agric. Food Chem.

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Changes in the activity and structure of alkaline phosphatase (ALP) and L-lactate dehydrogenase (LDH) were investigated after high pressure processing (HPP). HPP treatments (206-620 MPa for 6 and 12 min) were applied to ALP and LDH prepared in buffer, fat-free milk, and 2% fat milk. Enzyme activities were measured using enzymatic assays, and changes in structure were investigated using far-ultraviolet circular dichroism (CD) spectroscopy and dynamic light scattetering (DLS). Kinetic data indicated that the activity of ALP was not affected after 6 min of pressure treatments (206-620 MPa), regardless of the medium in which the enzyme was prepared. Increasing the processing time to 12 min did significantly reduce the activity of ALP at 620 MPa (P < 0.001). However, even the lowest HPP treatment of 206 MPa induced a reduction in LDH activity, and the course of reduction increased with HPP treatment until complete inactivation at 482, 515, and 620 MPa. CD data demonstrated a partial change in the secondary structure of ALP at 620 MPa, whereas the structure of LDH showed gradual denaturation after exposure at 206 MPa for 6 min, leading to a random coil structure at both 515 and 620 MPa. DLS results indicated aggregation of ALP only at HPP treatment of 206 MPa and not above and enzyme precipitation as well as aggregation at 345, 415, 482, and 515 MPa. The loss of LDH activity with increasing pressure and time treatment was due to the combined effects of denaturation and aggregation.

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Section: Resultsmentioning

confidence: 99%

Effect of High-Pressure Processing on Activity and Structure of Alkaline Phosphatase and Lactate Dehydrogenase in Buffer and Milk

Kouassi

Anantheswaran

Knabel

et al. 2007

J. Agric. Food Chem.

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“…A circular Dichroism experiment is commonly performed to analyze the structural changes in enzymes due to enzyme-inhibitor complex formation 76 . Generally, in CD analysis, the α-helix gives negative bands at 222 and 208 nm, β-sheet structures give a negative band at 210—220 nm, and the random coil has a characteristic negative band at 200 nm 77 . The far UV-CD spectrum of GIIA exhibited two distinct negative bands at 210 nm and 222 nm.…”

Section: Resultsmentioning

confidence: 99%

Group IIA secreted phospholipase A2 inhibition by elemolic acid as a function of anti-inflammatory activity

Krishnappa

Urs

Manjunatha

et al. 2022

Sci Rep

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Human group IIA secreted phospholipase A2 (GIIA) is a key enzyme in inflammatory reactions, worsening the condition of several chronic inflammatory diseases. The natural inhibitors of GIIA potentially block the production of inflammatory mediators. In the present study, elemolic acid, a triterpenoid from Boswellia serrata inhibited the GIIA enzyme in a concentration-dependent manner with IC50 value of 5.70 ± 0.02 µM. The mode of GIIA inhibition was studied by increasing the concentration of the substrate from 30 to 120 nM, and calcium from 2.5 to 15 mM, the level of inhibition was not changed. The inhibitor-enzyme interaction was examined by fluorimetry and Circular Dichroism (CD) studies; elemolic acid altered intrinsic fluorescence intensity and shifted far UV- CD spectra of GIIA enzyme, suggesting the direct interaction with GIIA. Elemolic acid neutralized the GIIA mediated indirect hemolytic activity from 94.5 to 9.8% and reduced GIIA induced mouse paw edema from 171.75 to 113.68%. Elemolic acid also reduced the hemorrhagic effect of GIIA along with Vipera russelii neurotoxic non-enzymatic peptide -VNTx-II (VR-HC-I). Thus, the elemolic acid has been proven as a potent inhibitor of GIIA enzyme and modulated the GIIA induced inflammatory response by in situ and in vivo methods.

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“…Although the chemical education literature includes numerous laboratory activities exploring the conformational stability of proteins (for recent examples, see Refs. 4–6), our search revealed no such activities exploring the resistance of proteins to proteolytic degradation. We have therefore designed a biochemistry laboratory exercise in which students explore protein degradation by the ubiquitin‐proteasome pathway in yeast.…”

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confidence: 93%

Exploring the ubiquitin‐proteasome protein degradation pathway in yeast

Will

McWatters

McQuade

2006

Biochem Molecular Bio Educ

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This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme ␤-galactosidase (␤-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce ␤-gal expression, cycloheximide is added to halt translation, and ␤-gal degradation is monitored by measuring enzyme activity as a function of time. Students observe that an N-Ile-␤-gal variant with an N-terminal isoleucine has a significantly lower stability than wild-type ␤-gal, whose N-terminal residue is methionine. This strong dependence of protein stability on the N-terminal residue is known as the "N-end rule." To corroborate the enzyme activity assay results, students perform denaturing protein electrophoresis and immunoblotting of lysates, observing that the time-dependent loss of enzyme activity is coincident with the disappearance of the ␤-gal protein.

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Influence of enzyme conformational changes on catalytic activity investigated by circular dichroism spectroscopy

Cited by 19 publications

References 15 publications

Effect of High-Pressure Processing on Activity and Structure of Alkaline Phosphatase and Lactate Dehydrogenase in Buffer and Milk

Effect of High-Pressure Processing on Activity and Structure of Alkaline Phosphatase and Lactate Dehydrogenase in Buffer and Milk

Group IIA secreted phospholipase A2 inhibition by elemolic acid as a function of anti-inflammatory activity

Exploring the ubiquitin‐proteasome protein degradation pathway in yeast

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