scholarly journals Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles

2004 ◽  
Vol 378 (3) ◽  
pp. 1059-1066 ◽  
Author(s):  
M. Soledad CELEJ ◽  
Mariana G. D'ANDREA ◽  
Patricia T. CAMPANA ◽  
Gerardo D. FIDELIO ◽  
M. Lucia BIANCONI
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Enzyme Stability ◽  
Catalytic Efficiency ◽  
Tryptophan Fluorescence ◽  
Enzyme Activation ◽  
Hexadecyltrimethylammonium Bromide ◽  
Negative Band ◽  
Cationic Micelles ◽  
Α Helix

The catalytic behaviour of α-CT (α-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme–micelle interaction leads to an increase in both the Vmax and the affinity for the substrate p-nitrophenyl acetate, indicating higher catalytic efficiency for bound α-CT. The bell-shaped profile of α-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in α-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the α-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in α-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of α-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased α-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm−1, corresponding to the α-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the β-sheet content in micelle-bound α-CT. Our data suggest that the higher catalytic efficiency of micelle-bound α-CT results from significant conformational changes.

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 Synergistic effects of functionally distinct substitutions in β-lactamase variants shed light on the evolution of bacterial drug resistance

2018 ◽  
Vol 293 (46) ◽  
pp. 17971-17984 ◽  
Author(s):  
Meha P. Patel ◽  
Liya Hu ◽  
Cameron A. Brown ◽  
Zhizeng Sun ◽  
Carolyn J. Adamski ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Enzyme Stability ◽  
Synergistic Effects ◽  
Catalytic Efficiency ◽  
Bacterial Drug Resistance ◽  
Natural Variant ◽  
Loop Conformation ◽  
Shed Light

The CTX-M β-lactamases have emerged as the most widespread extended-spectrum β-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103–106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103–106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 β-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a “switch” that toggles the conformations of the 103–106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M–like, cefotaximase-favorable conformation.

Binding to lipid membrane induces conformational changes in RPE65: implications for its isomerohydrolase activity

2011 ◽  
Vol 436 (3) ◽  
pp. 591-597 ◽  
Author(s):  
Olga Nikolaeva ◽  
Gennadiy Moiseyev ◽  
Karla K. Rodgers ◽  
Jian-xing Ma
Keyword(s):  
Fluorescence Quenching ◽  
Conformational Changes ◽  
Lipid Membrane ◽  
Membrane Binding ◽  
Tryptophan Fluorescence ◽  
Cd Spectroscopy ◽  
Visual Cycle ◽  
Α Helix ◽  
Structural Levels

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.

Conformational changes in bovine lactoferrin induced by slow or fast temperature increases

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Waleska D. Schwarcz ◽  
Lorena Carnelocce ◽  
Jerson L. Silva ◽  
Andréa C. Oliveira ◽  
Rafael B. Gonçalves
Keyword(s):  
Secondary Structure ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Heat Stability ◽  
Bovine Lactoferrin ◽  
Spectroscopic Techniques ◽  
Bioactive Component ◽  
Physiological Properties

Abstract Lactoferrin (LF) is an iron-binding protein present in several secreted substances, such as milk, and has broad antimicrobial and physiological properties. Because high temperatures may affect protein stability and its functional properties, we investigated the effect of heat on bovine LF structure and stability. The effects of temperatures used during the pasteurization process on LF and its relationship to protein functionality were studied. Conformational changes were monitored using spectroscopic techniques, such as circular dichroism (CD) and fluorescence spectroscopy. The CD data at 70°C showed that LF's secondary structure is drastically and irreversibly affected when the temperature is gradually increased. The same effect is observed when the temperature is gradually raised from 25°C to 105°C and changes are monitored by tryptophan fluorescence emission. We also verified the effects of simulating the pasteurization process; LF remained well structured during the entire process and this result was not time-dependent. Owing to preservation of the secondary structure with changes in the tertiary structure, we thus believe that pasteurization might cause LF to change into an intermediate partially folded state. A better understanding of heat stability is important for the use of LF as a bioactive component in food.

scholarly journals Molecular Dynamics Simulations Study of the Interactions between Human Dipeptidyl-Peptidase III and Two Substrates

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6492
Author(s):  
Shitao Zhang ◽  
Shuai Lv ◽  
Xueqi Fu ◽  
Lu Han ◽  
Weiwei Han ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Biological Activities ◽  
Catalytic Efficiency ◽  
Dipeptidyl Peptidase ◽  
Pain Modulation ◽  
Ang Ii ◽  
Α Helix ◽  
Dynamics Simulations

Human dipeptidyl-peptidase III (hDPP III) is capable of specifically cleaving dipeptides from the N-terminal of small peptides with biological activity such as angiotensin II (Ang II, DRVYIHPF), and participates in blood pressure regulation, pain modulation, and the development of cancers in human biological activities. In this study, 500 ns molecular dynamics simulations were performed on free-hDPP III (PDB code: 5E33), hDPP III–Ang II (PDB code: 5E2Q), and hDPP III–IVYPW (PDB code: 5E3C) to explore how these two peptides affect the catalytic efficiency of enzymes in terms of the binding mode and the conformational changes. Our results indicate that in the case of the hDPP III–Ang II complex, subsite S1 became small and hydrophobic, which might be propitious for the nucleophile to attack the substrate. The structures of the most stable conformations of the three systems revealed that Arg421-Lys423 could form an α-helix with the presence of Ang II, but only part of the α-helix was produced in hDPP III-IVYPW. As the hinge structure in hDPP III, the conformational changes that took place in the Arg421-Lys423 residue could lead to the changes in the shape and space of the catalytic subsites, which might allow water to function as a nucleophile to attack the substrate. Our results may provide new clues to enable the design of new inhibitors for hDPP III in the future.

scholarly journals Further structural and functional properties of mini‒lipoxygenase, an active fragment of soybean lipoxygenase-1

2004 ◽  
Vol 18 (2) ◽  
pp. 331-338 ◽  
Author(s):  
Mauro Maccarrone ◽  
Almerinda Di Venere ◽  
Guus van Zadelhoff ◽  
Giampiero Mei ◽  
Gerrit Veldink ◽  
...  
Keyword(s):  
Functional Properties ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Catalytic Efficiency ◽  
Soybean Lipoxygenase ◽  
Binding Studies ◽  
Irreversible Inhibitor ◽  
Structural And Functional Properties ◽  
The Stability ◽  
Induced Changes

Lipoxygenases (Loxs) form a homologous family of non-heme, non-sulfur iron containing lipid-peroxidizing enzymes, which catalyze the dioxygenation of polyunsaturated fatty acids to the corresponding hydroperoxy derivatives. Soybean lipoxygenase-1 (Lox-1) is widely used as a prototype for studying the structural and functional properties of lipoxygenases. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed “mini-Lox”, which shows enhanced catalytic efficiency and higher membrane binding ability than the native enzyme (M. Maccarrone, M.L. Salucci, G. van Zadelhoff, F. Malatesta, G. Veldink, J.F.G. Vliegenthart and A. Finazzi-Agrò,Biochemistry40(2001), 6819–6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride (GdHCl) and under high pressure by fluorescence and circular dichroism spectroscopy. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition. Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-8 anilinonaphtalene sulphonic acid binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by fluorescence techniques. In particular, eicosatetraynoic acid (ETYA), an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of ETYA. All these findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible and therefore less stable, that on the other hand is probably causing its higher catalytic efficiency.

scholarly journals The key amino acids of E protein involved in early flavivirus infection: viral entry

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Tao Hu ◽  
Zhen Wu ◽  
Shaoxiong Wu ◽  
Shun Chen ◽  
Anchun Cheng
Keyword(s):  
Amino Acids ◽  
Conformational Changes ◽  
Viral Entry ◽  
Envelope Protein ◽  
Cell Attachment ◽  
Envelope Proteins ◽  
Infection Process ◽  
Early Infection ◽  
Protein Amino Acids ◽  
Α Helix

AbstractFlaviviruses are enveloped viruses that infect multiple hosts. Envelope proteins are the outermost proteins in the structure of flaviviruses and mediate viral infection. Studies indicate that flaviviruses mainly use envelope proteins to bind to cell attachment receptors and endocytic receptors for the entry step. Here, we present current findings regarding key envelope protein amino acids that participate in the flavivirus early infection process. Among these sites, most are located in special positions of the protein structure, such as the α-helix in the stem region and the hinge region between domains I and II, motifs that potentially affect the interaction between different domains. Some of these sites are located in positions involved in conformational changes in envelope proteins. In summary, we summarize and discuss the key envelope protein residues that affect the entry process of flaviviruses, including the process of their discovery and the mechanisms that affect early infection.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

scholarly journals The effects of Ca2+ and Cd2+ on the secondary and tertiary structure of bovine testis calmodulin. A circular-dichroism study

1986 ◽  
Vol 238 (2) ◽  
pp. 485-490 ◽  
Author(s):  
S R Martin ◽  
P M Bayley
Keyword(s):  
Circular Dichroism ◽  
Conformational Changes ◽  
Metal Ion ◽  
Tertiary Structure ◽  
Thermal Unfolding ◽  
Apparent Effect ◽  
Thermally Induced ◽  
Circular Dichroïsm ◽  
Secondary And Tertiary Structure

Near-u.v. and far-u.v. c.d. spectra of bovine testis calmodulin and its tryptic fragments (TR1C, N-terminal half, residues 1-77, and TR2C, C-terminal half, residues 78-148) were recorded in metal-ion-free buffer and in the presence of saturating concentrations of Ca2+ or Cd2+ under a range of different solvent conditions. The results show the following: if there is any interaction between the N-terminal and C-terminal halves of calmodulin, it has not apparent effect on the secondary or tertiary structure of either half; the conformational changes induced by Ca2+ or Cd2+ are substantially greater in TR2C than they are in TR1C; the presence of Ca2+ or Cd2+ confers considerable stability with respect to urea-induced denaturation, both for the whole molecule and for either of the tryptic fragments; a thermally induced transition occurs in whole calmodulin at temperatures substantially below the temperature of major thermal unfolding, both in the presence and in the absence of added metal ion; the effects of Cd2+ are identical with those of Ca2+ under all conditions studied.

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