The catalase activity of diiron adenine deaminase

Journal Article · Thu Dec 01 00:00:00 EST 2011 · Protein Science

DOI:https://doi.org/10.1002/pro.748· OSTI ID:1044003

S, Kamat S; Swaminathan, S; Holmes-Hampton, G P; Bagaria, A; Kumaran, D; Tichy, S E; Gheyi, T; Zheng, X; Bain, K; Groshong, C; Emtage, S; Sauder, J M; Burley, S K; Lindahl, P A; Raushel, F M

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{sup 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{sub 4}. Inductively coupled plasma mass spectrometry and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 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{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup 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.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States)
• Sponsoring Organization: ELI LILLY & COMPANY
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 1044003
• Report Number(s): BNL-96556-2012-JA; TRN: US201214%%258
• Journal Information: Protein Science, Vol. 20, Issue 12; ISSN 0961-8368
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ADENINES
AMMONIA
CATALASE
CATALYSIS
DEAMINATION
ENZYMES
ESCHERICHIA COLI
HISTIDINE
HYDROXYL RADICALS
HYPOXANTHINE
INDUCTION
IRON
MASS SPECTROSCOPY
METHIONINE
MODIFICATIONS
MOESSBAUER EFFECT
MOLECULAR WEIGHT
MONOMERS
OXIDATION
PLASMA
PROTEINS
REDOX REACTIONS
REDUCTION
RESIDUES
VALENCE