Essential role of histidine 20 in the catalytic mechanism of Escherichia coli peptidyl-tRNA hydrolase.
The peptidyl-tRNA hydrolase (Pth) enzyme plays an essential role in recycling tRNA from peptidyl-tRNA that has prematurely dissociated from the ribosome. In this study of Escherichia coli Pth, the critical role of histidine 20 was investigated by site-directed mutagenesis, stopped-flow kinetic measurements, and chemical modification. The histidine residue at position 20 is known to play an important role in the hydrolysis reaction, but stopped-flow fluorescence measurements showed that, although the His20Asn Pth mutant enzyme was unable to hydrolyze the substrate, the enzyme retained the ability to bind peptidyl-tRNA. Chemical modification of Pth with diethyl pyrocarbonate (DEPC) showed that a residue, with a pK(a) value of 6.3, was essential for substrate hydrolysis and that the stoichiometry of inhibition was 0.70 +/- 0.06 mol of DEPC/mol of enzyme, indicating that modification of only a single residue by DEPC was responsible for the loss of activity. Parallel chemical modification studies with the His20Asn and Asp93Asn mutant enzymes showed that this essential residue was His20. These studies indicate that histidine 20 acts as the catalytic base in the hydrolysis of peptidyl-tRNA by Pth.
Goodall
Jonathan J
JJ
Basilea Pharmaceutica Ltd., Grenzacherstrasse 487, CH-4005 Basel, Switzerland.
Chen
Guo Jun
GJ
Page
Malcolm G P
MG
D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019811:Hydroxylamine / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D017354:Point Mutation; D013379:Substrate Specificity / Q000235:genetics
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C437127:O,O-diethylphosphorylcholine iodide; D010767:Phosphorylcholine; D019811:Hydroxylamine; D006639:Histidine; D001216:Asparagine; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase; D004047:Diethyl Pyrocarbonate