Understanding the flavoenzyme human augmenter of liver regeneration: biochemical and structural perspectives
Date
2013
Authors
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Publisher
University of Delaware
Abstract
Augmenter of liver regeneration (ALR) is a multifaceted protein with biological roles including, but not limited to, disulfide bond formation, mitochondrial fission and fusion, spermatogenesis and activation of the MAP kinase pathway. This dissertation explores kinetic, thermodynamic and structural aspects of this protein. The first three Chapters provide background information on disulfide bond formation, the ERV/ALR family of proteins and biological roles of ALR, respectively. In Chapter 4, we determine the rate-limiting step of this flavin-dependent enzyme during the oxidation of the model substrate dithiothreitol by molecular oxygen. We also determined the redox potential of the redox-active disulfide proximal to the FAD cofactor and gained new insight into the formation and stabilization of the charge-transfer intermediate. Chapters 5 and 6 investigate the structural and functional consequence of replacing sulfur with selenium in both a random (Chapter 5) and specific (Chapter 6) manner. The final Chapter briefly describes the structure of two active site mutants of ALR, C142S and C142A. The C142S mutation crystallized with the charge-transfer interaction intact while the C142A construct was susceptible to oxidation that resulted in a cysteine sulfinic acid at position 145.