title: Probing the catalytic mechanism of bovine pancreatic deoxyribonuclease I by chemical rescue.
abstract: Previous structural and mutational studies of bovine pancreatic deoxyribonuclease I (bpDNase I) have demonstrated that the active site His134 and His252 played critical roles in catalysis. In our present study, mutations of these two His residues to Gln, Ala or Gly reduced the DNase activity by a factor of four to five orders of magnitude. When imidazole or primary amines were added exogenously to the Ala or Gly mutants, the residual DNase activities were substantially increased by 60-120-fold. The rescue with imidazole was pH- and concentration-dependent. The pH-activity profiles showed nearly bell-shaped curves, with the maximum activity enhancement for H134A at pH 6.0 and that for H252A at pH 7.5. These findings indicated that the protonated form of imidazole was responsible for the rescue in H134A, and the unprotonated form was for that in H252A, prompting us to assign unambiguously the roles for His134 as a general acid, and His252 as a general base, in bpDNase I catalysis.
authors.affiliation: Institute of Biotechnology, College of Bioresources, National Ilan University, Ilan 26047, Taiwan. wjchen@niu.edu.tw
authors.forename: Wei-Jung
authors.initials: WJ
authors.lastname: Chen
authors.forename: Pei-Jun
authors.initials: PJ
authors.lastname: Lai
authors.forename: Yu-Shen
authors.initials: YS
authors.lastname: Lai
authors.forename: Po-Tsang
authors.initials: PT
authors.lastname: Huang
authors.forename: Chai-Ching
authors.initials: CC
authors.lastname: Lin
authors.forename: Ta-Hsiu
authors.initials: TH
authors.lastname: Liao
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D011485:Protein Binding
chemical_list: D004247:DNA; D003850:Deoxyribonuclease I