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Article

Analysis of the Link between Enzymatic Activity and Oligomeric State in AhpC, a Bacterial Peroxiredoxin^†^,^‡

Derek Parsonage,^§ Derek S. Youngblood, Ganapathy N. Sarma, Zachary A. Wood, P. Andrew Karplus,* and Leslie B. Poole*^§

Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, and Department of Biochemistry and Biophysics, Oregon State University, 2011 AG Life Sciences Building, Corvallis, Oregon 97331

Biochemistry, 2005, 44 (31), pp 10583–10592

DOI: 10.1021/bi050448i

Publication Date (Web): July 14, 2005

†

This study was supported by a grant from the National Institutes of Health (NIH) to L.B.P. with a subcontract to P.A.K. (RO1 GM50389) and an Established Investigatorship from the American Heart Association to L.B.P. (0140175N). D.S.Y. was partially supported by an undergraduate research fellowship funded by Howard Hughes Medical Institute Grant 57003741 awarded to Oregon State University (OSU) and an OSU Undergraduate Research, Innovation, Scholarship, Creativity award. This work was also made possible in part by Grant P30 ES00210 from the National Institute of Environmental Health Sciences, NIH.

‡

The coordinates and structure factors have been deposited in the Protein Data Bank as entries 1YF0, 1YF1, and 1YEX for T77I, T77V, and T77D, respectively, and 1YEP for the further refined wild-type AhpC coordinates (replacing entry 1KYG).

Wake Forest University School of Medicine.

Oregon State University.

To whom correspondence should be addressed. Telephone: (336) 716-6711. Fax: (336) 777-3242. E-mail: lbpoole@wfubmc.edu or karplusp@science.oregonstate.edu.

Abstract

Peroxiredoxins (Prxs) make up a ubiquitous class (proposed EC 1.11.1.15) of cysteine-dependent peroxidases with roles in oxidant protection and signal transduction. An intriguing biophysical property of typical 2-Cys Prxs is the redox-dependent modulation of their oligomeric state between decamers and dimers at physiological concentrations. The functional consequences of this linkage are unknown, but on the basis of structural considerations, we hypothesized that decamer-building (dimer−dimer) interactions serve to stabilize a loop that forms the peroxidatic active site. Here, we address this important issue by studying mutations of Thr77 at the decamer-building interface of AhpC from Salmonella typhimurium. Ultracentrifugation studies revealed that two of the substitutions (T77I and T77D) successfully disrupted the decamer, while the third (T77V) actually enhanced decamer stability. Crystal structures of the decameric forms of all three mutant proteins provide a rationale for their properties. A new assay allowed the first ever measurement of the true k_cat and K_m values of wild-type AhpC with H₂O₂, placing the catalytic efficiency at 4 × 10⁷ M^-¹ s^-¹. T77V had slightly higher activity than wild-type enzyme, and both T77I and T77D exhibited ca. 100-fold lower catalytic efficiency, indicating that the decameric structure is quite important for, but not essential to, activity. The interplay between decamer formation and active site loop dynamics is emphasized by a decreased susceptibility of T77I and T77D to peroxide-mediated inactivation, and by an increase in the crystallographic B-factors in the active site loop, rather than at the site of the mutation, in the T77D variant.