Catalytically Active Alkaline Molten Globular Enzyme: Effect of pH and Temperature on the Structural Integrity of 5-Aminolevulinate Synthase
Document Type
Article
Publication Date
2014
Keywords
Aminolevulinate synthase, Pyridoxal-5′-phosphate, Heme, Molten globule, Protein folding, Intrinsically disordered proteins
Digital Object Identifier (DOI)
https://doi.org/10.1016/j.bbapap.2014.09.013
Abstract
5-Aminolevulinate synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.
Was this content written or created while at USF?
Yes
Citation / Publisher Attribution
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, v. 1844, issue 12, p. 2145-2154
Scholar Commons Citation
Stojanovski, Bosko M.; Breydo, Leonid; Hunter, Gregory A.; Uversky, Vladimir N.; and Ferreira, Gloria C., "Catalytically Active Alkaline Molten Globular Enzyme: Effect of pH and Temperature on the Structural Integrity of 5-Aminolevulinate Synthase" (2014). Molecular Medicine Faculty Publications. 408.
https://digitalcommons.usf.edu/mme_facpub/408
