The effect of some osmolytes on the activity and stability of mushroom tyrosinase.
The thermodynamical stability and remained activity of mushroom tyrosinase (MT) from Agaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40 degrees C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obey the first order law. Inactivation rate constant (kinact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of kinact (0.7x10(-4) s-1) in comparison with their absence (2.5x10(-4) s-1). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodynamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.
Gheibi
N
N
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Saboury
A A
AA
Haghbeen
K
K
Moosavi-Movahedi
A A
AA
D000364:Agaricus / Q000201:enzymology*; D000596:Amino Acids / Q000494:pharmacology*; D001224:Aspartic Acid / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000494:pharmacology; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D009995:Osmosis; D010649:Phenylalanine / Q000494:pharmacology; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D014199:Trehalose / Q000494:pharmacology*
D000596:Amino Acids; D005656:Fungal Proteins; D001224:Aspartic Acid; D010649:Phenylalanine; D006639:Histidine; D014199:Trehalose; D014442:Monophenol Monooxygenase