Insight into stability of CotA laccase from the spore coat of Bacillus subtilis

The axial ligand of the catalytic mononuclear T1 copper site (Met502) of the CotA laccase was replaced by a leucine or phenylalanine residue to increase the redox potential of the enzyme. These mutations led to an increase in the redox potential by approx. 100 mV relative to the wild-type enzyme but the catalytic constant kcat in the mutant enzymes was severely compromised. This decrease in the catalytic efficiency was unexpected as the X-ray analysis of mutants has shown that replacement of methionine ligand did not lead to major structural changes in the geometry of the T1 centre or in the overall fold of the enzyme. However, the mutations have a profound impact on the thermodynamic stability of the enzyme. The fold of the enzyme has become unstable especially with the introduction of the larger phenylalanine residue and this instability should be related to the decrease in the catalytic efficiency. The instability of the fold for the mutant proteins resulted in the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states. Quenching of tryptophan fluorescence by acrylamide has further revealed that the intermediate state is partly unfolded.

Download Full-text

Stereochemical Change

Reaction Mechanisms of Inorganic and Organometallic Systems ◽

10.1093/oso/9780195301007.003.0006 ◽

2007 ◽

Author(s):

Robert B. Jordan

Keyword(s):

Structural Changes ◽

Axial Ligand ◽

General Area ◽

Ring Inversion ◽

Carbon Bond ◽

Carbon Carbon Bond ◽

The Subject ◽

Simple Systems ◽

Current Stage ◽

Metal Ligand

The kinetic and mechanistic aspects of this general area tend to be strongly dependent on the particular system. This makes general treatments and explanations impossible, at least at the current stage of understanding. Various aspects of this area have been summarized in some general reviews. Ligands bonded to a metal can undergo a number of structural changes that do not involve complete breaking of the metal-ligand bond(s). Such processes are the subject of the following sections. Many chelate ligands have conformers that can interconvert. For example, the conformers of ethylenediamine interchange by rotation about the carbon-carbon bond, as shown in the following structures: The Ha and Ha' protons are magnetically different from the Hb and Hb' protons, so their interconversion can, in principle, be studied by NMR. These protons may be referred to as exo and endo, respectively. In simple systems, their interconversion is too rapid (k >106 s-1) for this method. However, if there is some constraint (e.g., CH3 groups) or if the coordinating atoms are part of a larger chelate system, then interconversion is slow enough to be detected by NMR. In nonplanar Fe(III)- tetraphenylporphyrinates, the ring inversion rates vary widely, depending on the axial ligand and the substituents on the porphyrin.

Download Full-text

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

The Journal of Biochemistry ◽

10.1093/jb/mvaa083 ◽

2020 ◽

Vol 168 (5) ◽

pp. 557-567

Author(s):

Wanitcha Rachadech ◽

Yusuke Kato ◽

Rabab M Abou El-Magd ◽

Yuji Shishido ◽

Soo Hyeon Kim ◽

...

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Active Site ◽

Structural Changes ◽

Catalytic Efficiency ◽

Acid Oxidase ◽

Wild Type ◽

Amino Acid Oxidase ◽

Adenine Ring ◽

The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

Download Full-text

Structural changes without stable intermediate state in inelastic material. Part II. Applications to displacive and diffusional–displacive phase transformations, strain-induced chemical reactions and ductile fracture

International Journal of Plasticity ◽

10.1016/s0749-6419(99)00083-2 ◽

2000 ◽

Vol 16 (7-8) ◽

pp. 851-892 ◽

Cited By ~ 39

Author(s):

Valery I. Levitas

Keyword(s):

Ductile Fracture ◽

Phase Transformations ◽

Chemical Reactions ◽

Intermediate State ◽

Structural Changes ◽

Material Part ◽

Stable Intermediate ◽

Inelastic Material

Download Full-text

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Biologia ◽

10.2478/s11756-013-0290-3 ◽

2014 ◽

Vol 69 (1) ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Ping-Lin Ong ◽

Tzu-Ting Chuang ◽

Tzu-Fan Wang ◽

Long-Liu Lin

Keyword(s):

Bacillus Licheniformis ◽

Specific Activity ◽

Chloride Ion ◽

Tryptophan Fluorescence ◽

Host Cells ◽

Glycoside Hydrolase Family ◽

Chloride Binding ◽

Mutant Proteins ◽

Nickel Affinity Chromatography ◽

Intrinsic Tryptophan Fluorescence

AbstractBased on sequence alignment of selected Cl− dependent and independent glycoside hydrolase family 13 enzymes, two invariant residues (Arg201 and Asn347) and one tyrosine (Tyr365) that might be responsible for the binding of Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analyses. The mutant enzymes (R201Q/E/K, N327Q/D/K, and Y365A/R) and BlTreA were individually overexpressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified BlTreA and Y365A had a specific activity of 236.9 and 47.6 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With the exception of Y365A, all mutant enzymes did not have the ability to bind fluoride, chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of BlTreA. However, wild-type and mutant enzymes displayed a slight difference in the profiles of intrinsic tryptophan fluorescence. Collectively, these results clearly indicate that Arg201 and Agr327 residues might play an essential role in chloride binding of BlTreA.

Download Full-text

Defining the Role of the Axial Ligand of the Type 1 Copper Site in Amicyanin by Replacement of Methionine with Leucine

Biochemistry ◽

10.1021/bi900836h ◽

2009 ◽

Vol 48 (39) ◽

pp. 9174-9184 ◽

Cited By ~ 15

Author(s):

Moonsung Choi ◽

Narayanasami Sukumar ◽

Aimin Liu ◽

Victor L. Davidson

Keyword(s):

Axial Ligand ◽

Copper Site ◽

Type 1 Copper

Download Full-text

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

Biochemical Journal ◽

10.1042/bj20031536 ◽

2004 ◽

Vol 378 (3) ◽

pp. 1059-1066 ◽

Cited By ~ 65

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.

Download Full-text

Spectroscopic Studies and Electronic Structure Description of the High Potential Type 1 Copper Site in Fungal Laccase: Insight into the Effect of the Axial Ligand

Journal of the American Chemical Society ◽

10.1021/ja991087v ◽

1999 ◽

Vol 121 (30) ◽

pp. 7138-7149 ◽

Cited By ~ 66

Author(s):

Amy E. Palmer ◽

David W. Randall ◽

Feng Xu ◽

Edward I. Solomon

Keyword(s):

Electronic Structure ◽

Spectroscopic Studies ◽

Axial Ligand ◽

High Potential ◽

Fungal Laccase ◽

Copper Site ◽

Structure Description ◽

Type 1 Copper ◽

Insight Into

Download Full-text

Effect of cosolvents (polyols) on structural and foaming properties of soy protein isolate

Czech Journal of Food Sciences ◽

10.17221/35/2016-cjfs ◽

2017 ◽

Vol 35 (No. 1) ◽

pp. 57-66 ◽

Cited By ~ 3

Author(s):

Pan Mingzhe ◽

Meng Xianjun ◽

Jiang Lianzhou ◽

Yu Dianyu ◽

Liu Tianyi

Keyword(s):

Soy Protein ◽

Structural Changes ◽

Foam Stability ◽

Soy Protein Isolate ◽

Tryptophan Fluorescence ◽

Surface Hydrophobicity ◽

Cd Spectroscopy ◽

Protein Isolate ◽

Foaming Properties ◽

Intrinsic Tryptophan Fluorescence

Effect of polyols (mannitol, sorbitol, and xylitol) at three concentrations (5, 10, and 15% w/w) on the structure of soy protein isolates (SPI) was investigated. Changes in foaming properties of SPI were then examined with the addition of polyols at different concentrations. The interactions between SPI and polyols resulted in a substantial decrease in protein surface hydrophobicity and intrinsic tryptophan fluorescence intensity, along with the covering of tyrosine. Furthermore, circular dichroism (CD) spectroscopy of SPI suggested that a more ordered and compact conformation was induced by polyols. Consequently, these structural changes led to lower foamability of SPI. An increase in the viscosity of SPI suspension seemed to be advantageous for improving the foam stability of SPI.

Download Full-text