Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release

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• 作者：Erica J. Fratz ; Jerome Clayton ; Gregory A. Hunter ; Sarah Ducamp ; Leonid Breydo ; Vladimir N. Uversky ; Jean-Charles Deybach ; Laurent Gouya ; Herv茅 Puy ; Gloria C. Ferreira
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：September 15, 2015
• 年：2015
• 卷：54
• 期：36
• 页码：5617-5631
• 全文大小：655K
• ISSN：1520-4995

文摘

Regulation of 5-aminolevulinate synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5鈥?phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon binding of ALA to the XLPP variants demonstrated that the protein conformational transition step associated with product release was predominantly affected. Of relevance is the fact that XLPP could also be modeled in cell culture. We propose that (1) the XLPP mutations destabilize the succinyl-CoA-induced hALAS2 closed conformation and thus accelerate ALA release, (2) the extended C-terminus of wild-type mammalian ALAS2 provides a regulatory role that allows for allosteric modulation of activity, thereby controlling the rate of erythroid heme biosynthesis, and (3) this control is disrupted in XLPP, resulting in porphyrin accumulation.