The catalase activity of diiron adenine deaminase.

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

Catalytic mechanism and three-dimensional structure of adenine deaminase.

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k(cat) = 2.0 s(-1); k(cat)/K(m) = 2.5 × 10(3) M(-1) s(-1)). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn(2+) prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k(cat) and k(cat)/K(m) values of 200 s(-1) and 5 × 10(5) M(-1) s(-1), respectively. The apoenzyme was prepared and reconstituted with Fe(2+), Zn(2+), or Mn(2+). In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe(II)/Fe(II)]-ADE was oxidized to [Fe(III)/Fe(III)]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe(III)/Fe(III)]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Mössbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 Å resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate-limiting steps.