Aging-Associated Enzyme Human Clock-1: Substrate-Mediated Reduction of the Diiron Center for 5-Demethoxyubiquinone Hydroxylation
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Lippard_Aging-associated.pdf
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Author(s)
Lu, Tsai-Te
Lee, Seung Jae
Apfel, Ulf-Peter
Lippard, Stephen J.
Date Issued
February 2013
Journal
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.
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Author's final manuscript
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1021/bi301674p
