Identification of the hydrolytic moiety of the Neurospora plasma membrane H+-ATPase and demonstration of a phosphoryl-enzyme intermediate in its catalytic mechanism

Biochemistry ◽  
1980 ◽  
Vol 19 (13) ◽  
pp. 2931-2937 ◽  
Author(s):  
John B. Dame ◽  
Gene A. Scarborough
Keyword(s):  
Plasma Membrane ◽  
Catalytic Mechanism ◽  
Enzyme Intermediate

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.

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.

Water stress inhibits p-nitrophenyl phosphate hydrolysis activity of the plasma membrane H+-ATPase from soybean hypocotyls

2000 ◽  
Vol 27 (7) ◽  
pp. 717
Author(s):  
Quan-Sheng Qiu ◽  
Nan Zhang
Keyword(s):  
Plasma Membrane ◽  
Water Stress ◽  
Catalytic Mechanism ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Phosphatase Domain ◽  
Nitrophenyl Phosphate ◽  
Peg Treatment ◽  
Pnpp Hydrolysis

The influence of water stress on ATP and p-nitrophenyl phosphate (PNPP) hydrolysis by plasma mem-brane ATPases was investigated using plasma membrane vesicles purified from soybean hypocotyls by the sucrose gradient centrifugation method. Results showed that ATPase activity was reduced after 10% polyethylene glycol (PEG) 6000 treatment for 12 h. Water stress also moved the optimal pH from 6.5 to 7.0. A significant decrease in PNPP hydrolysis was observed under PEG treatment. The Km for PNPP hydrolysis was shifted from 2.3042 0.0009 to 2.5048 0.0346 mmol L –1 . Moreover, PNPP hydrolysis was more sensitive to vanadate after PEG treatment, while inhibition of ATP hydrolysis by hydroxylamine was not affected. Our experimental results indicated that water stress changed the catalytic mechanism of the plasma membrane H + -ATPase through affecting the dephosphorylation process catalysed by its phosphatase domain.

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