Replacement of catalytic histidine-195 of chloramphenicol acetyltransferase: evidence for a general base role for glutamate.
The imidazole N epsilon 2 of His-195 plays an essential part in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT), hydrogen bonding to and a abstracting a proton from the primary hydroxyl group of chloramphenicol. Replacement of His-195 by alanine or glutamine results in apparent decreases in kcat of (9 x 10(5)- and (3 x 10(5))-fold, respectively, whereas Km values for both substrates (chloramphenicol and acetyl-CoA) are similar to those of wild-type CAT. The structure of Gln-195 CAT has been solved at 2.5-A resolution and is largely isosteric with that of wild-type CAT. Substitution of His-195 by glutamate resulted in a (5 x 10(4))-fold decrease in kcat together with a 3-fold increase in the Km for chloramphenicol. Direct determination of binding constants for both substrates demonstrated that these substitutions result in only small decreases in the affinity of CAT for acetyl-CoA (Kd values increased 2- to 3-fold), whereas chloramphenicol Kd values are elevated 26-, 20-, and 53-fold for Ala-195 CAT, Gln-195 CAT and Glu-195 CAT, respectively. The pH dependence of kcat/Km yields apparent pKa values of 6.5 and 6.7 for Ala-195 CAT and Gln-195 CAT, respectively, which are very similar to that (6.6) determined for the ionization of His-195 in wild-type CAT. In contrast, the pH dependence of kcat/Km for Glu-195 CAT (pKa = 8.3) is very different from that of wild-type CAT.(ABSTRACT TRUNCATED AT 250 WORDS)
Lewendon
A
A
Department of Biochemistry, University of Leicester, U.K.
Murray
I A
IA
Shaw
W V
WV
Gibbs
M R
MR
Leslie
A G
AG
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D002384:Catalysis; D002701:Chloramphenicol / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003062:Codon; D005971:Glutamates / Q000737:chemistry*; D018698:Glutamic Acid; D005973:Glutamine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed
D003062:Codon; D005971:Glutamates; D005973:Glutamine; D018698:Glutamic Acid; D006639:Histidine; D002701:Chloramphenicol; D000105:Acetyl Coenzyme A; C047569:3-(bromoacetyl)chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D000409:Alanine