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 abstracting a proton from the primary hydroxyl group of chloramphenicol. Replacement of His-195 by alanine or glutamine results in apparent decreases in k(cat) of (9 x 10(5))- and (3 x 10(5))-fold, respectively, whereas K-m 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-Angstrom 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 k(cat) together with a 3-fold increase in the K-m 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 (K-d 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 k(cat)/K-m, yields apparent pK(2), 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 k(cat)/K-m for Glu-195 CAT (pK(2) = 8.3) is very different from that of wild-type CAT. Treatment with the affinity reagent 3-(bromoacetyl)chloramphenicol results in the rapid and complete inhibition of Ala-195 CAT, Gln-195 CAT, and wild-type enzyme whereas Glu-195 CAT retains 80% of its original activity. The results suggest that the activity detected in Ala-195 CAT and Gln-195 CAT arises from traces of wild-type CAT due to misincorporation of histidine. Since the properties of Glu-195 CAT cannot be explained by histidine misincorporation alone, it is likely that Glu-195 can replace His-195 as a general base, albeit inefficiently, in the mechanism of CAT. The anomalously high apparent pK(2) of Glu-195 probably reflects the nonpolar environment of residue-195 in CAT.