Elimination of a reactive thiol group from the active site of chloramphenicol acetyltransferase.
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.
Lewendon
A
A
Department of Biochemistry, University of Leicester, U.K.
Shaw
W V
WV
D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D014644:Genetic Variation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D013438:Sulfhydryl Compounds
D015345:Oligonucleotide Probes; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D015500:Chloramphenicol O-Acetyltransferase