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Title: 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{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 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{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential formore » catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} 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.« less
Authors:
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Publication Date:
OSTI Identifier:
1041938
Report Number(s):
BNL--97616-2012-JA
Journal ID: ISSN 0006-2960; BICHAW; TRN: US201212%%349
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry (Eaton); Journal Volume: 50; Journal Issue: 11
Research Org:
BROOKHAVEN NATIONAL LABORATORY (BNL)
Sponsoring Org:
USDOE SC OFFICE OF SCIENCE (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ADENINES; AMMONIA; CATALYSIS; CHEMICAL REACTIONS; CRYSTALLOGRAPHY; DEAMINATION; ELECTRON SPIN RESONANCE; ENZYMES; ESCHERICHIA COLI; FERRICYANIDES; HYPOXANTHINE; INACTIVATION; INDUCTION; IRON; ISOTOPE EFFECTS; MOESSBAUER EFFECT; MUTAGENESIS; RESIDUES; RESOLUTION; SOLVENTS; SPIN; VISCOSITY