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J. Biol. Chem., Vol. 280, Issue 24, 22651-22663, June 17, 2005

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The Met-Tyr-Trp Cross-link in Mycobacterium tuberculosis Catalase-peroxidase (KatG)

AUTOCATALYTIC FORMATION AND EFFECT ON ENZYME CATALYSIS AND SPECTROSCOPIC PROPERTIES^*

Reza A. Ghiladi, Giselle M. Knudsen, Katalin F. Medzihradszky, and Paul R. Ortiz de Montellano

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143

Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met²⁵⁵-Tyr²²⁹-Trp¹⁰⁷ cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared, making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA) or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this "cross-link-free" WT KatG with PAA revealed complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P·)Fe^III, where P· represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F) under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.

Received for publication, March 7, 2005 , and in revised form, April 14, 2005.

^* This work was supported by National Institutes of Health (NIH) Grant AI58524 (to R. A. G., an F32-Postdoctoral Fellowship), National Center for Research Resources Grants RR001614 and RR012961 (to K. F. M., to the UCSF Mass Spectrometry Facility, director A. L. Burlingame), and NIH Grants GM56531 and GM32488 (to P. R. O. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmaceutical Chemistry, University of California, San Francisco, 600 16th St., San Francisco, CA 94143-2280. Tel.: 415-476-2903; Fax: 415-502-4728; E-mail: ortiz{at}cgl.ucsf.edu.

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