The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

Abstract

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

Analysis of Hydrogen Bonding in Enzyme-Substrate Complexes of Chloramphenicol Acetyltransferase by Infrared Spectroscopy and Site-Directed Mutagenesis

“…Modeling studies produced a plausible model for the bound tetrahedral intermediate and predicted an important role for the hydroxyl of Ser-148 in stabilizing the putative oxyanion, a proposal subsequently confirmed by sitedirected mutagenesis and X-ray crystallography (Lewendon et al ,1990). The importance of His-195 had been predicted on the basis of sequence alignments and chemical modification studies (Kleanthous et al, 1985) and is supported by recent kinetic and mutagenic analyses (Lewendon & Shaw, 1993;Lewendon et al, 1994).…”

Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nε-acyltransferase involved in mycobactin biosynthesis

“…Furthermore, the participation of a histidine residue as a general base in an acyltransfer reaction is not without precedent. Serotonin acetyltransferase employs a pair of histidine residues that function redundantly through intervening water molecules [24] and chloramphenicol acetyltransferase takes advantage of a histidine base in a mechanistically similar reaction [25].…”

An in situ combinatorial methodology to synthesize and screen chemical probes

“…S16, ESI †). Alkylation of this histidine with methyl 4-nitrobenzenesulfonate 14 inhibited labeling of CAT, indicating that this residue might be modified by our probes. However, several of the sulfonyl fluorides that did not label CAT, including L9R5, adopt a similar pose.…”