Modification of a Methionine Residue near the Active Site of Chymotrypsin by p-Nitrophenyl Bromoacetyl-α-aminoisobutyrate*

Biochemistry ◽  
1965 ◽  
Vol 4 (3) ◽  
pp. 377-386 ◽  
Author(s):  
William B. Lawson ◽  
Hans-Josef Schramm
Keyword(s):  
Active Site ◽  
Methionine Residue

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 Elimination of a reactive thiol group from the active site of chloramphenicol acetyltransferase

1990 ◽  
Vol 272 (2) ◽  
pp. 499-504 ◽  
Author(s):  
A Lewendon ◽  
W V Shaw
Keyword(s):  
Active Site ◽  
Thiol Group ◽  
Chloramphenicol Acetyltransferase ◽  
Side Chain ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Hydrophobic Amino Acid ◽  
Nitrobenzoic Acid ◽  
Methionine Residue

1. The type III variant of chloramphenicol acetyltransferase (CATIII) is resistant to inactivation by ionizable modifying reagents such as 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and iodoacetate, whereas it is sensitive to inhibition by similar but uncharged reagents, including 4,4′-dithiodipyridine, methyl methanethiolsulphonate (MMTS) and iodoacetamide. The target for these thiol-modifying reagents has been postulated to be Cys-31. This residue is situated within a part of the chloramphenicol-binding site formed largely from the side chains of hydrophobic amino acid residues, which might be expected to discriminate against the access of ionized ligands to Cys-31. 2. The substitution of Cys-31 by alanine, serine, threonine or methionine yields an enzyme that is resistant to inactivation by thiol-specific reagents. Replacement of Cys-31 by alanine, serine or threonine results in increased Km values for chloramphenicol with only small changes in kcat.. In contrast, the Cys-31----Met substitution mainly affects kcat. values. Although the kcat. for chloramphenicol acetylation is decreased 13-fold compared with wild-type CAT, the kcat. for the acetyl-CoA hydrolysis reaction, which occurs in the absence of chloramphenicol, is increased 2.7-fold. 3. MMTS modification of cysteine residues results in an adduct (-CH2-S-S-CH3) that is structurally similar to the side chain of a methionine residue (-CH2-CH2-S-CH3). The kinetic properties of MMTS-modified CATIII closely resemble those of [Met31]CAT.

Oxidation of a methionine residue in subtilisin-type proteinases by the hydrogen peroxide/borate system — an active site-directed reaction

1988 ◽  
Vol 952 ◽  
pp. 20-26 ◽  
Author(s):  
Gert Hausdorf ◽  
Kathrin Krüger ◽  
Gabriele Küttner ◽  
Hermann-Georg Holzhütter ◽  
Cornelius Frömmel ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Active Site ◽  
Methionine Residue
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