The Role of Zinc Atoms in Nuclease P1

paper,3) we showed that the removal of 3 zinc atoms from the enzyme molecule resulted in complete inactivation and disruption of the helical structure with insolubilization of the enzyme. Such a zinc-deprived enzyme could no longer react with anti-nuclease P1 antiserum.4) However, the role of the individual zinc atoms could not be defined. In this paper, we studied the role of the individual zinc atoms in the structure and function of nuclease Pi by selective removal of each of three zinc atoms from the enzyme. Highly purified nuclease P1 preparation1) was used. The enzyme concentration was determined spectrophotometrically, assuming that E11%cm m at 28Onm was 18.4. The removal of zinc atoms from the enzyme molecule was carried out as follows: nuclease P1 (200 ƒÊg/ml) was incubated in M/35 Veromal buffer, pH 5.3, containing l •~107' •`l•~10-1 M ethylene diamine tetraacetate (EDTA) at 37•Ž for 30 min. Nucleolytic activity on RNA and 3'AMP-dephosphorylating activity was assayed as described previously.1) Substrate solutions and buffer solution were passed through a column of chelating resin, Diaion CR 10 (Mitsubishi Kasei Kogyo Co., Ltd.). CD measurements in the far-ultraviolet region of 200•`250 nm were performed with a Japan Spectroscopic Model J-500 A with a quartz cell of 0.1 cm path length at about 0.09% of the enzyme concentration at room temperature. The reduced mean residue ellipticities [ƒÆ] were calculated from the relationship [ƒÆ]=M/101c., where ƒÆ is the observed ellipticity in deg, and M is the mean residue molecular weight, 114. Unfolding profile was assessed by measuring the dichronic signal at 222 nm which is a sensitive parameter of the polypeptide back bone conformation.5) Zinc was determined by atomic adsorption with a Varian Techtron atomic adsorption spectrophotometer Model 1100 following dialysis of the sample for removal of EDTA.