scholarly journals Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site*

1997 ◽  
Vol 322 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Ernst BAUSE ◽  
Marko WESEMANN ◽  
Achim BARTOSCHEK ◽  
Wilhelm BREUER
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Epoxy Group ◽  
Covalent Attachment ◽  
Current View ◽  
Double Labelling ◽  
Acceptor Site ◽  
Hydrogen Bond Acceptor ◽  
Suicide Inactivation

Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asn-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-α,γ-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own ‘suicide’ inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation [Bause, Breuer and Peters (1995) Biochem. J. 312, 979Ő985].

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.

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.

scholarly journals Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Martino L. Di Salvo ◽  
J. Neel Scarsdale ◽  
Galina Kazanina ◽  
Roberto Contestabile ◽  
Verne Schirch ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Serine Hydroxymethyltransferase ◽  
Active Site Residue ◽  
Wild Type ◽  
Hydroxymethyl Group ◽  
New Energy ◽  
Mutant Forms

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stablegem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion ofgem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and theε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

scholarly journals Probing the role of the conserved residue Glu166 in a class A β-lactamase using neutron and X-ray protein crystallography

2020 ◽  
Vol 76 (2) ◽  
pp. 118-123
Author(s):  
Patricia S. Langan ◽  
Brendan Sullivan ◽  
Kevin L. Weiss ◽  
Leighton Coates
Keyword(s):  
Water Molecule ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Drug Binding ◽  
Bacterial Strains ◽  
Structural Explanation ◽  
X Ray ◽  
Class A ◽  
Kinetic Rates

The amino-acid sequence of the Toho-1 β-lactamase contains several conserved residues in the active site, including Ser70, Lys73, Ser130 and Glu166, some of which coordinate a catalytic water molecule. This catalytic water molecule is essential in the acylation and deacylation parts of the reaction mechanism through which Toho-1 inactivates specific antibiotics and provides resistance to its expressing bacterial strains. To investigate the function of Glu166 in the acylation part of the catalytic mechanism, neutron and X-ray crystallographic studies were performed on a Glu166Gln mutant. The structure of this class A β-lactamase mutant provides several insights into its previously reported reduced drug-binding kinetic rates. A joint refinement of both X-ray and neutron diffraction data was used to study the effects of the Glu166Gln mutation on the active site of Toho-1. This structure reveals that while the Glu166Gln mutation has a somewhat limited impact on the positions of the conserved amino acids within the active site, it displaces the catalytic water molecule from the active site. These subtle changes offer a structural explanation for the previously observed decreases in the binding of non-β-lactam inhibitors such as the recently developed diazobicyclooctane inhibitor avibactam.

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.

Reaction of threonine synthase with the substrate analogue 2-amino-5-phosphonopentanoate: implications into the proton transfer at the active site

2019 ◽  
Vol 167 (4) ◽  
pp. 357-364
Author(s):  
Yasuhiro Machida ◽  
Takeshi Murakawa ◽  
Akiko Sakai ◽  
Mitsuo Shoji ◽  
Yasuteru Shigeta ◽  
...  
Keyword(s):  
Catalytic Reaction ◽  
Active Site ◽  
Catalytic Mechanism ◽  
The Other ◽  
Amino Group ◽  
Threonine Synthase ◽  
Spectral Changes ◽  
Proton Migration ◽  
Insight Into

Abstract Threonine synthase catalyses the conversion of O-phospho-l-homoserine and a water molecule to l-threonine and has the most complex catalytic mechanism among the pyridoxal 5′-phosphate-dependent enzymes. In order to study the less-characterized earlier stage of the catalytic reaction, we studied the reaction of threonine synthase with 2-amino-5-phosphonopentanoate, which stops the catalytic reaction at the enamine intermediate. The global kinetic analysis of the triphasic spectral changes showed that, in addition to the theoretically expected pathway, the carbanion is rapidly reprotonated at Cα to form an aldimine distinct from the external aldimine directly formed from the Michaelis complex. The Kd for the binding of inhibitor to the enzyme decreased with increasing pH, showing that the 2-amino-group-unprotonated form of the ligand binds to the enzyme. On the other hand, the rate constants for the proton migration steps within the active site are independent of the solvent pH, indicating that protons are shared by the active dissociative groups and are not exchanged with the solvent during the course of catalysis. This gives an insight into the role of the phosphate group of the substrate, which may increase the basicity of the ε-amino group of the catalytic lysine residue in the active site.

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