The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase.
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.
Lewendon
A
A
Department of Biochemistry, University of Leicester, U.K.
Shaw
W V
WV
D001557:Benzenesulfonates / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D015500:Chloramphenicol O-Acetyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics
D001557:Benzenesulfonates; D006639:Histidine; C010233:methyl-4-nitrobenzenesulfonate; D015500:Chloramphenicol O-Acetyltransferase; D003545:Cysteine; D007460:Iodoacetamide