Nuclear Magnetic Resonance Studies of Manganese Binding of Rat Liver Arginase

Abstract

Abstract Magnetic resonance studies of the activation of arginase (l-arginine amidinohydrolase, EC 3.5.3.1) with manganese have been performed with nuclear magnetic resonance. The results showed that 4 moles of manganese were bound per mole of fully activated arginase. However, the binding affinities of the metal ions to the enzyme were not identical; 50% of the Mn2+ was strongly bound and could not be removed without considerable irreversible loss of activity and solubility of the enzyme. Various treatments such as dialysis, electrophoresis, or chelating of the Mn2+ by an excess of complexing agents resulted in enzyme preparations that retained about 50% of their original activity; full activity could be recovered in these samples by the addition of Mn2+. The amount of tightly bound manganese estimated by quantitative electron spin resonance measurements after denaturation of the enzyme with 1.5 n HCl showed that the dialyzed enzyme, with a residual catalytic activity of 50%, contained 2 moles of manganese per mole of arginase, suggesting that the enzyme existed then in the form of an E-Mn2 complex. Measurements of the effect of the manganese in the E-Mn2 complex on the longitudinal nuclear relaxation rate of water protons at pH 7.5 and 34° yielded an enhancement of 8.0 and a binding constant (K1) whose lower limit is 3 x 107 m-1. The activation step of arginase from E-Mn2 ⇌ E-Mn4, which results in a restoration of 100% of the catalytic activity, was studied kinetically at increasing manganese concentrations and at various temperatures and pH. By titration of the enzyme with manganese and vice versa, the formation of the E-Mn4 complex was followed by measurements of the proton relaxation rate, which yielded an enhancement of 6 to 8 at pH 7.5 and 34°. The binding constant for the reversibly removable Mn2+ was calculated to be K2 = 2 ± 1 x 104 m-1. The addition of arginine (substrate) or or nithine (competitive inhibitor) did not further increase the enhancement; rather, it slightly lowered it, which proves that primarily a binary complex between arginase and manganese is formed.