Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I.
Bovine pancreatic deoxyribonuclease I (DNase I) is a well characterised endonuclease which cleaves double-stranded DNA to yield 5' phosphorylated polynucleotides. Co-crystal structures of DNase I with two different oligonucleotides have revealed the presence of several residues (R9, E78, H134, D168, D212 and H252) close to the scissile phosphate. The roles that these amino acids play in the catalytic mechanism have been investigated using site-directed mutagenesis. The following variants were used: R9A, E78T, H134Q, D168S, D212S and H252Q. The kinetics of all six mutants with both DNA and a small chromophoric substrate, thymidine-3',5'-di(p-nitrophenyl)-phosphate, were studied. Only R9A and E78T showed any significant turnover of the two substrates. D168S, H134Q, D212S and H252Q showed vanishingly low activities towards DNA and no detectable activity with thymidine-3',5'-di(p-nitrophenyl)-phosphate. These results demonstrate that H134, D168, D212 and H252 play a critical role in the catalytic mechanism. It is suggested that H134 and H252 (which are hydrogen-bonded to E78 and D212, respectively) provided general acid and general base catalysis. DNase I also requires Mg2+ and E39 has been identified as a ligand for this metal ion. We propose that D168 serves as a ligand for a second Mg2+, and thus DNase I, uses a two metal-ion hydrolytic mechanism. Both magnesium ions are used to supply electrophilic catalysis. Role assignment is based on the mutagenesis results, structural information, homologies between DNase I from different species and a comparison with exonuclease III. However, it is still not feasible to unequivocally assign a particular catalytic role to each amino acid/metal ion.
Jones
S J
SJ
Department of Biochemistry and Genetics, University of Newcastle, Newcastle upon Tyne, UK.
Worrall
A F
AF
Connolly
B A
BA
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology; D017354:Point Mutation; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism
D013942:Thymine Nucleotides; D004247:DNA; D003850:Deoxyribonuclease I