Defects in heme biosynthesis can lead to microcytic anemias, iron homeostasis malfunction (e.g., hemochromatosis) and consequently to heart diseases. 5-Aminolevulinate synthase (ALAS) catalyzes the first, and regulatory, step of heme biosynthesis in mammals. The presently incurable X-linked sideroblastic anemia (XLSA) and the newly identified X-linked dominant protoporphyria (XLDPP) are human inherited microcytic anemias due to defects in erythroid ALAS (ALAS2). Previously, we demonstrated that the rate-limiting step of the ALAS-catalyzed reaction is product release, which we postulated to be controlled by an interconversion between two protein conformations. We further postulated that binding of the succinyl-CoA substrate modulates the ALAS conformational changes. Here we propose to unravel if gain-of-function mutations associated with ALAS2 C-terminal deletions in XLDPP hinder the binding of succinyl-CoA and lessen the extent of protein conformational changes concurrent with ALA release. The three general hypotheses are: 1) succinyl-CoA stabilizes the closed conformation by 'gating' the active site loop, 2) the active site loop controls the catalytic rate constant by limiting the rate of ALA release, and 3) C-terminal deletions in human ALAS2 (XLDPP) enhance the rate of ALA synthesis by destabilizing the active site loop, while eliminating hydrophobic and electrostatic interactions that would otherwise stabilize the closed conformation. To test these hypotheses, the following Specific Aims will be addressed: 1. Examine the role(s) of key active site residues in defining the closed conformation and mechanism of ALAS. 2. Characterize the reaction mechanism of the 6 'XLDPP variants' and establish whether their ALAS hyperactivity correlates with the opening of the active site loop and ALA release. A combination of experimental approaches, ranging from construction of ALAS and 'XLDPP' variants to the characterization of succinyl-CoA coordination, substrate binding and kinetic and structural properties, will provide interpretations at a molecular level of heme biosynthesis and iron homeostasis and provide the molecular basis for XLDPP. Significantly, compounds that stabilize the ALAS closed conformation should provide an easy-reach therapy for XLDPP. In addition, results may lead to in-situ overproduction of photosensitizers and new modalities of photodynamic therapy for atherosclerosis and cardiovascular disorders.
|Effective start/end date||07/01/2010 → 06/30/2012|