13 research outputs found
Resonance Raman Investigation of the Interaction of Thromboxane Synthase with Substrate Analogues â€
Role of the Oxyferrous Heme Intermediate and Distal Side Adduct Radical in the Catalase Activity of Mycobacterium tuberculosis KatG Revealed by the W107F Mutant*
Catalase-peroxidase (KatG) is essential in Mycobacterium
tuberculosis for oxidative stress management and activation of the
antitubercular pro-drug isoniazid. The role of a unique distal side adduct
found in KatG enzymes, involving linked side chains of residues
Met255, Tyr229, and Trp107 (MYW), in the
unusual catalase activity of KatG is addressed here and in our companion paper
(Suarez, J., Ranguelova, K., Jarzecki, A. A., Manzerova, J., Krymov, V., Zhao,
X., Yu, S., Metlitsky, L., Gerfen, G. J., and Magliozzo, R. S. (2009) J.
Biol. Chem. 284, in press). The KatG[W107F] mutant exhibited severely
reduced catalase activity yet normal peroxidase activity, and as isolated
contains more abundant 6-coordinate heme in high spin and low spin forms
compared with the wild-type enzyme. Most interestingly, oxyferrous heme is
also found in the purified enzyme. Oxyferrous KatG[W107F] was prepared by
photolysis in air of the carbonyl enzyme or was generated using hydrogen
peroxide decayed with a t½ of 2 days compared with
6 min for wild-type protein. The stability of oxyenyzme was modestly enhanced
in KatG[Y229F] but was not affected in KatG[M255A]. Optical stopped-flow
experiments showed rapid formation of Compound I in KatG[W107F] and facile
formation of oxyferrous heme in the presence of micromolar hydrogen peroxide.
An analysis of the relationships between catalase activity, stability of
oxyferrous enzyme, and a proposed MYW adduct radical is presented. The loss of
catalase function is assigned to the loss of the MYW adduct radical and
structural changes that lead to greatly enhanced stability of oxyenzyme, an
intermediate of the catalase cycle of native enzyme