scholarly journals Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZβ-galactosidase of Escherichia coli by β-d-galactopyranosylmethyl-p-nitrophenyltriazene

1978 ◽  
Vol 175 (2) ◽  
pp. 525-538 ◽  
Author(s):  
M L Sinnott ◽  
P J Smith
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Sequence Data ◽  
Free Enzyme ◽  
Irreversible Inhibitor ◽  
Methionine Residue ◽  
Complete Inactivation ◽  
Carbonium Ion ◽  
Affinity Labelling ◽  
Field Desorption Mass Spectrometry

1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500.

scholarly journals Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase

1989 ◽  
Vol 261 (2) ◽  
pp. 431-435 ◽  
Author(s):  
H G Nimmo ◽  
F Douglas ◽  
C Kleanthous ◽  
D G Campbell ◽  
C MacKintosh
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Isocitrate Lyase ◽  
Cysteine Residue ◽  
Order Process ◽  
Reactive Group ◽  
Site Isolation ◽  
First Order ◽  
Complete Inactivation ◽  
Ph Range

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues

scholarly journals Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

2021 ◽  
Vol 22 (9) ◽  
pp. 4769
Author(s):  
Pablo Maturana ◽  
María S. Orellana ◽  
Sixto M. Herrera ◽  
Ignacio Martínez ◽  
Maximiliano Figueroa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Conformational Dynamics ◽  
High Specificity ◽  
Arginine Decarboxylase ◽  
Space Groups ◽  
X Ray Crystallography ◽  
High Dynamics ◽  
Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

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