Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei.
The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +/- 0.29 M for intrinsic fluorescence and 4.90 +/- 0.31 M for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 +/- 0.010 microM and a Hill coefficient H of 1.00 +/- 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DeltaGH2OD increased from 6.83 +/- 0.43 to 8.42 +/- 0.55 kcal.mol-1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+.
Schwerdtfeger
R M
RM
Technical University Hamburg-Harburg, Institute of Biotechnology, Department of Technical Microbiology, Hamburg, Germany.
Chiaraluce
R
R
Consalvi
V
V
Scandurra
R
R
Antranikian
G
G
D020106:Acrylamide / Q000494:pharmacology; D019843:Archaeal Proteins / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004795:Enzyme Stability; D005453:Fluorescence; D006026:Glycoside Hydrolases / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D019711:Pyrococcus / Q000201:enzymology*; D013816:Thermodynamics
D019843:Archaeal Proteins; D002135:Calcium-Binding Proteins; D020106:Acrylamide; D006026:Glycoside Hydrolases; C100181:pullulanase; D019791:Guanidine; D002118:Calcium