Identification of a Phosphorylated Enzyme Intermediate in the Catalytic Mechanism for Selenophosphate Synthetase

1997 ◽  
Vol 119 (28) ◽  
pp. 6684-6685 ◽  
Author(s):  
Leisha S. Mullins ◽  
Suk-Bong Hong ◽  
Grant E. Gibson ◽  
Heidi Walker ◽  
Thressa C. Stadtman ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Selenophosphate Synthetase ◽  
Enzyme Intermediate

scholarly journals Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jaeyong Lee ◽  
Liam J. Worrall ◽  
Marija Vuckovic ◽  
Federico I. Rosell ◽  
Francesco Gentile ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Bound State ◽  
Crystallographic Structure ◽  
Wild Type ◽  
Site Mutation ◽  
Main Protease ◽  
Dimerization Interface ◽  
Enzyme Intermediate

AbstractSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes the disease COVID-19, produces replicase polyproteins 1a and 1ab that contain, respectively, 11 or 16 nonstructural proteins (nsp). Nsp5 is the main protease (Mpro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for subsequent viral assembly and maturation. We have determined X-ray crystallographic structures of this cysteine protease in its wild-type free active site state at 1.8 Å resolution, in its acyl-enzyme intermediate state with the native C-terminal autocleavage sequence at 1.95 Å resolution and in its product bound state at 2.0 Å resolution by employing an active site mutation (C145A). We characterize the stereochemical features of the acyl-enzyme intermediate including critical hydrogen bonding distances underlying catalysis in the Cys/His dyad and oxyanion hole. We also identify a highly ordered water molecule in a position compatible for a role as the deacylating nucleophile in the catalytic mechanism and characterize the binding groove conformational changes and dimerization interface that occur upon formation of the acyl-enzyme. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for future antiviral therapeutic development including revised molecular docking strategies based on Mpro inhibition.

scholarly journals Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Venu Gopal Vandavasi ◽  
Patricia S. Langan ◽  
Kevin L. Weiss ◽  
Jerry M. Parks ◽  
Jonathan B. Cooper ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Active Site Residues ◽  
Hydrogen Atoms ◽  
Content Type ◽  
X Ray Crystallography ◽  
General Base ◽  
Class A ◽  
Extended Spectrum ◽  
Enzyme Intermediate

ABSTRACT The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

scholarly journals Structural Insights into the Catalytic Mechanism of Escherichia coli Selenophosphate Synthetase

2011 ◽  
Vol 194 (2) ◽  
pp. 499-508 ◽  
Author(s):  
N. Noinaj ◽  
R. Wattanasak ◽  
D.-Y. Lee ◽  
J. L. Wally ◽  
G. Piszczek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Mechanism ◽  
Selenophosphate Synthetase ◽  
Structural Insights

The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli

2004 ◽  
Vol 82 (11) ◽  
pp. 1581-1588 ◽  
Author(s):  
Andrew G Watts ◽  
Stephen G Withers
Keyword(s):  
Sialic Acid ◽  
Catalytic Mechanism ◽  
Competitive Inhibitor ◽  
Enzymatic Process ◽  
Trypanosoma Rangeli ◽  
Available N ◽  
Processing Enzymes ◽  
Acid Processing ◽  
Catalytic Mechanisms ◽  
Enzyme Intermediate

Sialyl hydrolases, trans-sialidases, and sialyl transferases are biologically important enzymes that are responsible for the incorporation and removal of sialic acid residues, which decorate many cell surface glycocongugates. Two fluorinated sialic acid derivatives have been synthesized as mechanism-based inactivators, to probe the catalytic mechanisms through which sialidases and trans-sialidases operate. Both compounds are known to be covalent inactivators of a trans-sialidase from Trypanosoma cruzi. Here, 3-fluorosialosyl fluoride has been found to covalently label the catalytic nucleophile of a sialidase from T. rangeli, and the residue involved is shown to be Tyr346 within the sequence DENSGYSSVL. This is the first demonstration that sialidases operate through a covalent glycosyl-enzyme intermediate, strongly suggesting a common catalytic mechanism amongst all members of the sialidase superfamily. CMP-3-fluoro sialic acid is a competitive inhibitor of sialyl transferases and was synthesized via a two-step enzymatic process from commercially available N-acetyl mannosamine, 3-fluoropyruvic acid, and cytidine triphosphate in around 84% yield.Key words: sialidase, mechanism, labeling, nucleophile, inhibitor.

scholarly journals Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane

1995 ◽  
Vol 309 (1) ◽  
pp. 113-118 ◽  
Author(s):  
L Hernandez ◽  
J Arrieta ◽  
C Menendez ◽  
R Vazquez ◽  
A Coego ◽  
...  
Keyword(s):  
Sugar Cane ◽  
Catalytic Mechanism ◽  
Polymerase Activity ◽  
High Yield ◽  
Sucrose Hydrolysis ◽  
Acetobacter Diazotrophicus ◽  
Ping Pong ◽  
Order Of Magnitude ◽  
Enzyme Intermediate

Acetobacter diazotrophicus, a nitrogen-fixing bacterium associated with sugar cane, secretes a levansucrase (sucrose-2,6-beta-D-fructan 6-beta-D-fructosyltransferase; EC 2.4.1.10). This enzyme is constitutively expressed and represents more than 70% of the total proteins secreted by strain SRT4. The purified protein consists of a single 58 kDa polypeptide with an isoelectric point of 5.5. Its activity is optimal at pH 5.0. It catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), fructan (polymerase reaction) and sucrose (oligofructoside synthesis). In vivo the polymerase activity leads to synthesis of a high-molecular-mass fructan of the levan type. A. diazotrophicus levansucrase catalyses transfructosylation via a Ping Pong mechanism involving the formation of a transient fructosyl-enzyme intermediate. The catalytic mechanism is very similar to that of Bacillus subtilis levansucrase. The kinetic parameters of the two enzymes are of the same order of magnitude. The main difference between the two enzyme specificities is the high yield of oligofructoside, particularly 1-kestotriose and kestotetraose, accumulated by A. diazotrophicus levansucrase during sucrose transformation. We discuss the hypothesis that these catalytic features may serve the different biological functions of each enzyme.

Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis

2021 ◽  
Vol 77 (2) ◽  
pp. 205-216
Author(s):  
Laetitia Collet ◽  
Corinne Vander Wauven ◽  
Yamina Oudjama ◽  
Moreno Galleni ◽  
Raphael Dutoit
Keyword(s):  
Catalytic Mechanism ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Enzymatic Assays ◽  
Conserved Residues ◽  
Displacement Mechanism ◽  
Significant Movement ◽  
Binding Cleft ◽  
Hydrolase Family ◽  
Enzyme Intermediate

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double-displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo-1,4-β-glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double-displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the −1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH-A.

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