title: The unfolding pathways of the native and molten globule states of 5-aminolevulinate synthase.
abstract: In this communication, we report the equilibrium and kinetic properties of the unfolding pathways of the native (pH 7.5) and alkaline molten globule (pH 10.5) states of the pyridoxal 5'-phosphate (PLP)-dependent enzyme 5-aminolevulinate synthase (ALAS). The stability of the molten globule state is adversely affected by thermal- and guanidine hydrochloride (GuHCl)-induced denaturation, and the equilibrium unfolding pathways, irrespective of pH, cannot be described with simple two-state models. Rapid kinetic measurements, in the presence of denaturing GuHCl concentrations, reveal that at pH 10.5, the rate of ALAS denaturation is 3 times faster than at pH 7.5. From pH jump experiments, comparable rates for the denaturation of the tertiary structure and PLP-microenvironment were discerned, indicating that the catalytic active site geometry strongly depends on the stable tertiary structural organization. Lastly, we demonstrate that partially folded ALAS tends to self-associate into higher oligomeric species at moderate GuHCl concentrations.
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA. Electronic address: stojanovskibm@slu.edu.
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; USF Health Byrd Alzheimer's Research Institute, 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; USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
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. Electronic address: gferreir@health.usf.edu.
authors.forename: Gloria C
authors.initials: GC
authors.lastname: Ferreira
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000737:chemistry* / Q000648:ultrastructure*; D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
chemical_list: D011732:Pyridoxal Phosphate; D000624:5-Aminolevulinate Synthetase; C480079:ALAS2 protein, human
keywords: 5-Aminolevulinate synthase; Heme; Molten globule; Protein unfolding; Pyridoxal phosphate