Site-directed mutagenesis of phosphate-contacting amino acids of bovine pancreatic deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (DNase I) is an endonuclease which cl eaves double-stranded DNA. Cocrystal structures of DNase I with oligonucleo tides have revealed interactions between the side chains of several amino a cids (N74, R111, N170, S206, T207, and Y211) and the DNA phosphates. The ef fects these interactions have on enzyme catalysis and DNA hydrolysis select ivity have been investigated by site-directed mutagenesis. Mutations to R11 1, N170, T207, and Y211 severely compromised activity toward both DNA and a small chromophoric substrate. A hydrogen bond between R111 (which interact s with the phosphate immediately 5' to the cutting site) and the essential amino acid H134 is probably required to maintain this histidine in the corr ect orientation for efficient hydrolysis. Both T207 and Y211 bind to the ph osphate immediately 3' to the cleavage site. Additionally, T207 is involved in binding an essential, structural, calcium ion, and Y211 is the nearest neighbor to D212, a critical catalytic residue. N170 interacts with the sci ssile phosphate and appears to play a direct role in the catalytic mechanis m. The mutation N74D, which interacts with a phosphate twice removed from t he scissile group, strongly reduced DNA hydrolysis. However, a comparison o f DNase I variants from several species suggests that certain amino acids, which allow interaction with phosphates (positively charged or hydrogen bon ding), are tolerated. S206, which binds to a DNA phosphate two positions aw ay from the cleavage site, appears to play a relatively unimportant role. N one of the enzyme variants, including a triple mutation in which N74, R111, and Y211 were altered, affected DNA hydrolysis selectivity. This suggests that phosphate binding residues play no role in the selection of DNA substr ates.