Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate.
The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.
Lewendon
A
A
Department of Biochemistry, University of Leicester, U.K.
Murray
I A
IA
Kleanthous
C
C
Cullis
P M
PM
Shaw
W V
WV
D000409:Alanine; D001120:Arginine; D001216:Asparagine; D001224:Aspartic Acid; D001665:Binding Sites; D002701:Chloramphenicol / Q000031:analogs & derivatives / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000031:analogs & derivatives; D006358:Hot Temperature; D006860:Hydrogen Bonding; D007700:Kinetics; D009154:Mutation; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013439:Sulfhydryl Reagents; C026814:3-carboxymethylhistidine; D001224:Aspartic Acid; D006639:Histidine; D002701:Chloramphenicol; D001216:Asparagine; D001120:Arginine; C047569:3-(bromoacetyl)chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D003545:Cysteine; D000409:Alanine