title: Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.
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 pH2.0, 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 (pH10.5 and pH9.5/37°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 420nm to 330nm 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 pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°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.
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Bosko M
authors.initials: BM
authors.lastname: Stojanovski
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Leonid
authors.initials: L
authors.lastname: Breydo
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Hunter
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA; USF Health Byrd Alzheimer's Research Institute, University of South Florida, Tampa, FL 33612, USA; Biology Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia; Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA; Department of Chemistry, University of South Florida, Tampa, FL 33612, USA. Electronic address: gferreir@health.usf.edu.
authors.forename: Gloria C
authors.initials: GC
authors.lastname: Ferreira
keywords: Aminolevulinate synthase; Heme; Intrinsically disordered proteins; Molten globule; Protein folding; Pyridoxal-5′phosphate