Paramagnetic probes for carbamoyl-phosphate synthetase: metal ion binding studies and preparation of nitroxide spin-labeled derivatives.
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abstract
Carbamoyl-phosphate synthetase from Escherichia coli has been shown to require a free divalent cation for activity in addition to the metal ions needed to complex the nucleotides used in the reaction. Activity with Mg2+, Mn2+, Co2+, Cd2+, and Zn2+ has been demonstrated, but no activity could be shown with Ca2+ or Ni2+ in the overall reaction. A double-reciprocal plot of velocity vs. [MgATP] at various [Mg2+]free is equilibrium-ordered with Mg2+ binding before MgATP. Thus, the free metal ion site must be populated for catalysis to occur and therefore free Mg2+ is an absolute requirement for enzymatic activity and not just a positive allosteric effector. The dissociation constant for free Mg2+ from this kinetic study is 4.2 0.7 mM. Metal ion binding studies to the free metal ion site were conducted by using electron paramagnetic resonance (EPR) techniques. These studies show one tight Mn2+ site with a dissociation constant of 35 3 M and some nonspecific weaker sites. By competition experiments with Mn2+, the binding constants at pH 7.5 for the other metal ions are the following: Mg2+, 5.2 mM; Co2+, 270 M; Ni2+, 960 M; Zn2+, 22 M;Cd2+, 220 M; Ca2+, 9.4 mM; Gd3+, 140 M. Since the dissociation constant for Mg2+ from the enzyme is in reasonable agreement in both the kinetic and binding experiments, they most likely refer to the same site. The substitution-inert ATP analogues Cr3+ATP and Co3+-(NH3)4ATP are competitive inhibitors vs. MgATP at pH 6.5. Three different sulfhydryl groups on carbamoyl-phosphate synthetase have been derivatized with 3-maleimido-2, 2, 5, 5-tetramethylpyrrolidinyl-1-oxy. Two of the sulfhydryl are located on the large subunit, and one is located on the small glutamine binding subunit. The EPR spectra of the two spin-labels located on the large subunit are insensitive to additions of substrates or allosteric modifiers. The mobility of the spin-label on the glutamine subunit is restricted upon the binding of glutamine and enhanced upon the binding of MgATP. The effect by MgATP shows that binding of substrates at one subunit can cause conformational changes at the other subunit. By use of the method of Leigh [Leigh, J. S. (1970) J. Chem. Phys. 52, 2680], it has been shown that all three nitroxide groups are < 20 away from the Mn2+ and MnATP sites. 1979, American Chemical Society. All rights reserved.
