Thermodynamic characterization of the palm tree Roystonea regia peroxidase stability.
The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization.
Zamorano
Laura S
LS
Departamento de Química Física, Facultad de Ciencias Químicas, Universidad de Salamanca, Salamanca, Spain.
Pina
David G
DG
Arellano
Juan B
JB
Bursakov
Sergey A
SA
Zhadan
Andrey P
AP
Calvete
Juan J
JJ
Sanz
Libia
L
Nielsen
Peter R
PR
Villar
Enrique
E
Gavel
Olga
O
Roig
Manuel G
MG
Watanabe
Leandra
L
Polikarpov
Igor
I
Shnyrov
Valery L
VL
D028023:Arecaceae / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D009195:Peroxidase / Q000737:chemistry*; D017510:Protein Folding; D055503:Protein Multimerization; D013816:Thermodynamics
D009195:Peroxidase