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J. Biol. Chem., Vol. 279, Issue 19, 19977-19986, May 7, 2004

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Probing the Active Site Loop Motif of Murine Ferrochelatase by Random Mutagenesis^*

Zhen Shi and Gloria C. Ferreira¶||

From the Department of Biochemistry and Molecular Biology, College of Medicine and ^¶H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612

Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis. We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction. Functional substitutions in the 10 consecutive loop positions Gln²⁴⁸–Leu²⁵⁷ were identified by genetic complementation in Escherichia coli strain vis. Lys²⁵⁰, Val²⁵¹, Pro²⁵³, Val²⁵⁴, and Pro²⁵⁵ tolerated a variety of replacements including single substitutions and contained low informational content. Gln²⁴⁸, Ser²⁴⁹, Gly²⁵², Trp²⁵⁶, and Leu²⁵⁷ possessed high informational content, since permissible replacements were limited and only observed in multiply substituted mutants. Selected active loop variants exhibited k_cat values comparable with or higher than that of wild-type murine ferrochelatase. The K_m values for porphyrin increased, except for the single mutant V251L. Other than a moderate increase observed in the triple mutant S249A/K250Q/V251C, the K_m values for Fe²⁺ were lowered. The k_cat/K_m for porphyrin remained largely unchanged, with the exception of a 10-fold reduction in the triple mutant K250M/V251L/W256Y. The k_cat/K_m for Fe²⁺ was improved. Molecular modeling of these active loop variants indicated that loop mutations resulted in alterations of the active site architecture. However, despite the plasticity of the loop primary structure, the relative spatial positioning of the loop in the active site appeared to be maintained in functional variants, supporting a role for the loop in ferrochelatase function.

Received for publication, December 17, 2003 , and in revised form, January 30, 2004.

^* This work was supported by American Cancer Society Grant RSG-96-05106-TBE and American Heart Association/Florida Affiliate Grant 0051240B. 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.

Recipient of an American Heart Association/Florida Division Predoctoral Fellowship.

^|| To whom correspondence should be addressed. Tel.: 813-974-5797; Fax: 813-974-0504; E-mail: gferreir{at}hsc.usf.edu.

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