Evidence That Proline Focuses Movement of the Floppy Loop of Arylalkylamine N-Acetyltransferase (EC 2.3.1.87)*
- Jiri Pavlicek‡,
- Steven L. Coon‡,
- Surajit Ganguly‡,1,
- Joan L. Weller‡,
- Sergio A. Hassan§,
- Dan L. Sackett¶ and
- David C. Klein‡,2
- ‡Section of Neuroendocrinology, Program on Developmental Endocrinology and Genetics and ¶Section on Cell Biophysics, Laboratory of Integrative and Medical Biophysics, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health and §Center for Molecular Modeling, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892
- ↵2 To whom correspondence should be addressed: National Institutes of Health 49-6A82, Bethesda, MD. Tel.: 301-496-6915; Fax: 301-480-3526; E-mail: kleind{at}mail.nih.gov.
Abstract
Arylalkylamine N-acetyltransferase (AANAT) catalyzes the N-acetylation of serotonin, the penultimate step in the synthesis of melatonin. Pineal AANAT activity increases at night in all vertebrates, resulting in increased melatonin production. This increases circulating levels of melatonin, thereby providing a hormonal signal of darkness. Kinetic and structural analysis of AANAT has determined that one element is floppy. This element, termed Loop 1, is one of three loops that comprise the arylalkylamine binding pocket. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL, and the enzyme became highly active. Here we focused on the functional importance of the CPL tripeptide and found that activity was markedly reduced when it was absent. Moreover, increasing the local flexibility of this tripeptide region by P64G and P64A mutations had the counterintuitive effect of reducing activity and reducing the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that these mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis and do not become immobilized by intramolecular interactions.
Footnotes
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↵* This work was supported, in whole or in part, by the NIH Intramural Research Program through the NICHD, National Institutes of Health and the Center for Information Technology. 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.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–4.
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↵1 Present address: The Centre for Genomic Application, Phase-III Okhla Industrial Estate, New Delhi, India 110020.
- Received January 23, 2008.
- Revision received March 14, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
