Aging-Associated Enzyme Human Clock-1: Substrate-Mediated Reduction of the Diiron Center for 5-Demethoxyubiquinone Hydroxylation
Author(s)
Lu, Tsai-Te; Lee, Seung Jae; Apfel, Ulf-Peter; Lippard, Stephen J.
DownloadLippard_Aging-associated.pdf (1.701Mb)
PUBLISHER_POLICY
Publisher Policy
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Terms of use
Metadata
Show full item recordAbstract
The mitochondrial membrane-bound enzyme Clock-1 (CLK-1) extends the average longevity of mice and Caenorhabditis elegans, as demonstrated for Δclk-1 constructs for both organisms. Such an apparent impact on aging and the presence of a carboxylate-bridged diiron center in the enzyme inspired this work. We expressed a soluble human CLK-1 (hCLK-1) fusion protein with an N-terminal immunoglobulin binding domain of protein G (GB1). Inclusion of the solubility tag allowed for thorough characterization of the carboxylate-bridged diiron active site of the resulting GB1-hCLK-1 by spectroscopic and kinetic methods. Both UV–visible and Mössbauer experiments provide unambiguous evidence that GB1-hCLK-1 functions as a 5-demethoxyubiquinone-hydroxylase, utilizing its carboxylate-bridged diiron center. The binding of DMQ[subscript n] (n = 0 or 2) to GB1-hCLK-1 mediates reduction of the diiron center by nicotinamide adenine dinucleotide (NADH) and initiates O[subscript 2] activation for subsequent DMQ hydroxylation. Deployment of DMQ to mediate reduction of the diiron center in GB1-hCLK-1 improves substrate specificity and diminishes consumption of NADH that is uncoupled from substrate oxidation. Both V[subscript max] and [k[subscript cat] over K[subscript M]] for DMQ hydroxylation increase when DMQ[subscript 0] is replaced by DMQ[subscript 2] as the substrate, which demonstrates that an isoprenoid side chain enhances enzymatic hydroxylation and improves catalytic efficiency.
Date issued
2013-02Department
Massachusetts Institute of Technology. Department of ChemistryJournal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
Lu, Tsai-Te, Seung Jae Lee, Ulf-Peter Apfel, and Stephen J. Lippard. “Aging-Associated Enzyme Human Clock-1: Substrate-Mediated Reduction of the Diiron Center for 5-Demethoxyubiquinone Hydroxylation.” Biochemistry 52, no. 13 (April 2, 2013): 2236–2244.
Version: Author's final manuscript
ISSN
0006-2960
1520-4995