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 pr oposed general base mechanism of chloramphenicol acetyltransferase (CA T), hydrogen bonding to and abstracting a proton from the primary hydr oxyl group of chloramphenicol. Replacement of His-195 by alanine or gl utamine 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 resoluti on and is largely isosteric with that of wild-type CAT. Substitution o f His-195 by glutamate resulted in a (5 x 10(4))-fold decrease in k(ca t) together with a 3-fold increase in the K-m for chloramphenicol. Dir ect determination of binding constants for both substrates demonstrate d that these substitutions result in only small decreases in the affin ity 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-1 95 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 con trast, the pH dependence of k(cat)/K-m for Glu-195 CAT (pK(2) = 8.3) i s very different from that of wild-type CAT. Treatment with the affini ty reagent 3-(bromoacetyl)chloramphenicol results in the rapid and com plete inhibition of Ala-195 CAT, Gln-195 CAT, and wild-type enzyme whe reas Glu-195 CAT retains 80% of its original activity. The results sug gest that the activity detected in Ala-195 CAT and Gln-195 CAT arises from traces of wild-type CAT due to misincorporation of histidine. Sin ce the properties of Glu-195 CAT cannot be explained by histidine misi ncorporation alone, it is likely that Glu-195 can replace His-195 as a general base, albeit inefficiently, in the mechanism of CAT. The anom alously high apparent pK(2) of Glu-195 probably reflects the nonpolar environment of residue-195 in CAT.