Hydrolysis of p-nitrophenyl phenylphosphonate catalysed by bovine pancreatic deoxyribonuclease.
The ability of bovine pancreatic DNAase to hydrolyse the synthetic substrate p-nitrophenyl phenylphosphonate (NPPP) is intrinsic and is not due to the contamination of the DNAase preparation by nonspecific phosphodiesterases because the activities of DNA and NPPP hydrolysis are co-eluted from a DEAE-cellulose column with use of the Ca2+-affinity elution method and because the two activities are decreased simultaneously when the purified enzyme is treated with Cu2+/iodoacetate, an active-site-labelling agent for DNAase. NPPP hydrolysis is facilitated by the metal ion-DNAase. At relatively high Na+ concentrations, where the metal ion-DNA interaction is weak, DNA hydrolysis is also facilitated by the metal ion-DNAase. With NPPP as substrate the Michaelis constants are Km 3.7 mM for Mn2+ and Km 49 mM for Mg2+ in 0.2 M-Tris/HCl buffer, pH 7.2. Ca2+ competes with Mn2+, with Ki 64 mM. Free Cu2+ ions non-competitively inhibit DNAase-catalysed DNA or NPPP hydrolysis in the presence of Mn2+ or Mg2+ and the inhibition is not relieved by Ca2+. The affinity of Cu2+ for free DNAase is higher than that for Mn2+-DNAase. Mn2+ is not bound to DNAase via a simple ionic interaction, as Mn2+ remains bound in the presence of relatively high Na+ concentrations and induces a near-u.v. difference absorption spectrum. The kinetics of NPPP hydrolysis catalysed by Mn2+-DNAase are sigmoidal. From the Hill equation, h = 2.0 is obtained, suggesting that more than two NPPP molecules are bound per molecule of DNAase with a certain amount of co-operativity. Because DNAase in solution is a monomer with a single catalytic site, the multiple NPPP molecules on a single protein molecule are probably in one location, resulting in a co-operative interaction that may resemble that in the stacked base-pairs of double-helical DNA.
Liao
T H
TH
Department of Biochemistry, National Taiwan University/College of Medicine, Taipei, Republic of China.
Hsieh
J C
JC
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D003300:Copper / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006868:Hydrolysis; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009596:Nitrophenols / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology
D007461:Iodoacetates; D009596:Nitrophenols; C058183:4-nitrophenyl phenylphosphonate; D008345:Manganese; D012965:Sodium Chloride; D003300:Copper; D003851:Deoxyribonucleases; D008274:Magnesium; D002118:Calcium; D019807:Iodoacetic Acid