Purification and Properties of Nicotinamide Deamidase from Flavobacterium peregrinum

Abstract

Abstract Nicotinamide deamidase from Flavobacterium peregrinum was purified about 210-fold with the use of protamine and MnCl2 treatments, acetone fractionation, ammonium sulfate fractionation, Sephadex G-200, and Cm-Sephadex column chromatography. The optimum pH of the purified nicotinamide deamidase is 6.5, and the Km value for nicotinamide is 0.2 µm. The molecular weight was estimated by means of gel filtration to be 48,000. The enzyme activity of the crude preparation was increased by 2.5-fold by addition of Mn2+ at a concentration of 10 mm followed by incubation at 37° for 30 min. The purified enzyme preparation was inactivated by dialysis in the absence of Mn2+ and no recovery of enzyme activity was possible even when Mn2+ or Mn2+ plus cysteine was added to the reaction mixture. When the enzyme was dialyzed overnight in the presence of Mn2+, almost all of the enzyme activity was retained. A dialysis of the enzyme against 0.01 m maleate buffer (pH 6.5) containing 5 µm HgCl2 resulted in complete retention of enzyme activity when 50 mm cysteine was added to the reaction mixture. CuSO4 and SnCl2 also showed effects similar to that of HgCl2, but their potency for enzyme stability was small. Mg2+, Ni2+, Zn2+, and Fe2+ did not show any effect on enzyme activity. All the nicotinamide analogues having a trivalent nitrogen in the pyridine ring showed a competitive inhibition of nicotinamide deamidation, whereas compounds having a tetravalent nitrogen seemed to have no effect. From these results the structure of the enzyme-metal-substrate complex was proposed as [see PDF for equation]