Computational investigations on the catalytic mechanism of maleate isomerase: the role of the active site cysteine residues

2014 ◽  
Vol 16 (24) ◽  
pp. 12462-12474 ◽  
Author(s):  
Hisham M. Dokainish ◽  
Bogdan F. Ion ◽  
James W. Gauld
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
The State ◽  
Cysteine Residues ◽  
Active Site Cysteine ◽  
Computational Enzymology

Multiscale computational enzymology provides key insights into the state of the substrate-bound active site and roles of its cysteinyl residues.

Characterization of anEscherichia coliSulfite Oxidase Homologue Reveals the Role of a Conserved Active Site Cysteine in Assembly and Function†

Biochemistry ◽  
2005 ◽  
Vol 44 (30) ◽  
pp. 10339-10348 ◽  
Author(s):  
Stephen J. Brokx ◽  
Richard A. Rothery ◽  
Guijin Zhang ◽  
Derek P. Ng ◽  
Joel H. Weiner
Keyword(s):  
Active Site ◽  
Active Site Cysteine ◽  
And Function

scholarly journals GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

2009 ◽  
Vol 420 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Helge C. Dorfmueller ◽  
Vladimir S. Borodkin ◽  
Marianne Schimpl ◽  
Daan M. F. van Aalten
Keyword(s):  
Active Site ◽  
Cell Biology ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Acetyl Group ◽  
Nanomolar Range ◽  
Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

On the Reactivity and Ionization of the Active Site Cysteine Residues ofEscherichia coliThioredoxin†

Biochemistry ◽  
1996 ◽  
Vol 35 (25) ◽  
pp. 8342-8353 ◽  
Author(s):  
Nobuyuki Takahashi ◽  
Thomas E. Creighton
Keyword(s):  
Active Site ◽  
Cysteine Residues ◽  
Active Site Cysteine

scholarly journals Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

2006 ◽  
Vol 401 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Paul A. O'Farrell ◽  
Leemor Joshua-Tor
Keyword(s):  
Water Molecule ◽  
Active Site ◽  
Bound Water ◽  
Active Site Residues ◽  
Site Structure ◽  
Active Site Cysteine ◽  
C Terminus ◽  
Bleomycin Hydrolase ◽  
Active Site Mutants

Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes.

Identification of Active Site Cysteine Residues that Function as General Bases:  Diaminopimelate Epimerase

2000 ◽  
Vol 122 (25) ◽  
pp. 6122-6123 ◽  
Author(s):  
Carolyn W. Koo ◽  
Andrew Sutherland ◽  
John C. Vederas ◽  
John S. Blanchard
Keyword(s):  
Active Site ◽  
Cysteine Residues ◽  
Active Site Cysteine

scholarly journals Active site alanine substitutions can convert deubiquitinating enzymes into avid ubiquitin-binding domains

2017 ◽  
Author(s):  
Marie Morrow ◽  
Michael Morgan ◽  
Marcello Clerici ◽  
Katerina Growkova ◽  
Ming Yan ◽  
...  
Keyword(s):  
Active Site ◽  
Tight Binding ◽  
Dominant Negative ◽  
Structural Basis ◽  
Deubiquitinating Enzymes ◽  
Active Site Cysteine ◽  
Binding Domains ◽  
Common Strategy ◽  
Catalytic Cysteine

ABSTRACTA common strategy for studying the biological role of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

scholarly journals Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1130
Author(s):  
Timo Meyer ◽  
Nadine Zumbrägel ◽  
Christina Geerds ◽  
Harald Gröger ◽  
Hartmut H. Niemann
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Catalytic Mechanism ◽  
Building Blocks ◽  
Secondary Amines ◽  
Hydrophobic Amino Acids ◽  
High Degree

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

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