Chemical modification of bovine pancreatic deoxyribonuclease with phenylglyoxal--the involvement of Arg-9 and Arg-41 in substrate binding.
The inactivation of bovine pancreatic DNase by phenylglyoxal exhibits pseudo-first-order and pH-dependent kinetics. At 13.2 mM phenylglyoxal and 25 degrees C, the half-life of DNase is 8 min at pH 8.0 and 2 h at pH 6.7. Calcium, which binds to DNase, does not protect against or facilitate the reaction of DNase with the reagent. However, due to DNA-DNase interaction the half-life of DNase is approx. doubled in the presence of 0.2% (w/v) DNA. Modified DNase has apparently lost its ability to interact with DNA since it elutes behind native DNase on a Sepharose 4B column developed with buffer containing DNA. Complete inactivation of the enzyme is achieved when approx. 4 of the 12 arginines in DNase are modified at pH 6.7. The identification of the radioactive peptides, isolated from the proteolytic digest of [7-14C]phenylglyoxal-treated DNase, showed the four modified arginines to be Arg-9, -27, -30 and -41. Based on the data from dual labeling experiments using a mixture of DNase modified (without DNA protection) by radioactive phenylglyoxal and DNase modified (with DNA protection) by cold phenylglyoxal, it is concluded that Arg-27 and Arg-30 are essentially un-protected by DNA while Arg-9 and Arg-41 are protected part of the time. This conclusion agrees with the proposed substrate binding site in the three-dimensional structure of DNase (Suck, D., Lahm, A. and Oefner, C. (1988) Nature 332, 464-468) where Arg-9 and Arg-41 are among the residues responsible for interaction with DNA.
Liao
T H
TH
Department of Biochemistry, College of Medicine, National Taiwan University, Taipei, China.
Ho
H C
HC
Abe
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002417:Cattle; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010658:Phenylglyoxal / Q000378:metabolism / Q000494:pharmacology*
D001120:Arginine; D003851:Deoxyribonucleases; D010658:Phenylglyoxal