Catalytic mechanism and three-dimensional structure of adenine deaminase.
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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 Mssbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 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.
author list (cited authors)
Kamat, S. S., Bagaria, A., Kumaran, D., Holmes-Hampton, G. P., Fan, H., Sali, A., ... Raushel, F. M.
complete list of authors
Kamat, Siddhesh S||Bagaria, Ashima||Kumaran, Desigan||Holmes-Hampton, Gregory P||Fan, Hao||Sali, Andrej||Sauder, J Michael||Burley, Stephen K||Lindahl, Paul A||Swaminathan, Subramanyam||Raushel, Frank M