Topographical differences in the active site region of α-chymotrypsin, subtilisin Novo, and subtilisin Carlsberg. Mapping the aromatic binding site by inhibitors (virtual substrates)

Biochemistry ◽  
1974 ◽  
Vol 13 (2) ◽  
pp. 266-277 ◽  
Author(s):  
Hans R. Bosshard ◽  
Arieh Berger
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Subtilisin Carlsberg ◽  
Active Site Region

scholarly journals Activity of linked HIV-1 proteinase dimers containing mutations in the active site region

1996 ◽  
Vol 9 (11) ◽  
pp. 997-1003 ◽  
Author(s):  
Péter Bagossi ◽  
Yin-Shyun E. Cheng ◽  
Stephen Oroszlan ◽  
József Tözsér
Keyword(s):  
Active Site ◽  
Active Site Region ◽  
Hiv 1

The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3)

1998 ◽  
Vol 17 (7) ◽  
pp. 699-712 ◽  
Author(s):  
Dennis E. Epps ◽  
Roger A. Poorman ◽  
Gary L. Petzold ◽  
Christopher W. Stuchly ◽  
Alice L. Laborde ◽  
...  
Keyword(s):  
Binding Site ◽  
Fluorescent Probes ◽  
Active Site

scholarly journals A crystallographic study of the Toho-1 β-lactamase acylation mechanism

2014 ◽  
Vol 70 (a1) ◽  
pp. C1207-C1207
Author(s):  
Leighton Coates
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Ligand Complex ◽  
Transition State Analog ◽  
Hydrogen Bonding Interactions ◽  
Active Site Region ◽  
Neutron Crystallography

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

scholarly journals Digital dissection of arsenate reductase enzyme from an arsenic hyperccumulating fern Pteris vittata

2016 ◽  
Author(s):  
Zarrin Basharat ◽  
Deeba Noreen Baig ◽  
Azra Yasmin
Keyword(s):  
Neural Network ◽  
Active Site ◽  
Tertiary Structure ◽  
Pteris Vittata ◽  
Arsenate Reductase ◽  
Dynamics Simulation ◽  
Reduction Mechanism ◽  
Arsenic Reduction ◽  
Secondary And Tertiary Structure ◽  
Active Site Region

Action of arsenate reductase is crucial for the survival of an organism in arsenic polluted area. Pteris vittata, also known as Chinese ladder brake, was the first identified arsenic hyperaccumulating fern with the capability to convert [As(V)] to arsenite [As(III)]. This study aims at sequence analysis of the most important protein of the arsenic reduction mechanism in this specie. Phosphorylation potential of the protein along with possible interplay of phosphorylation with O-β-GlcNAcylation was predicted using neural network based webservers. Secondary and tertiary structure of arsenate reductase was then analysed. Active site region of the protein comprised a rhodanese-like domain. Cursory dynamics simulation revealed that folds remained conserved in the rhodanese main but variations were observed in the structure in other regions. This information sheds light on the various characteristics of the protein and may be useful to enzymologists working on the improvement of its traits for arsenic reduction.

scholarly journals Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

2004 ◽  
Vol 382 (2) ◽  
pp. 751-757 ◽  
Author(s):  
Pakorn WINAYANUWATTIKUN ◽  
Albert J. KETTERMAN
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Mechanism ◽  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Anopheles Dirus ◽  
Steady State Kinetics ◽  
Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

Probing the specificity of the S1 binding site of subtilisin Carlsberg with boronic acids

1991 ◽  
Vol 176 (1) ◽  
pp. 401-405 ◽  
Author(s):  
Thomas H. Keller ◽  
Peter Seufer-Wasserthal ◽  
J.Bryan Jones
Keyword(s):  
Binding Site ◽  
Boronic Acids ◽  
Subtilisin Carlsberg
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