Stability, refolding and Ca 2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei

• Published in: European journal of biochemistry Vol. 264; no. 2; pp. 479 - 487
• Main Authors: Schwerdtfeger, Ruth M, Chiaraluce, Roberta, Consalvi, Valerio, Scandurra, Roberto, Antranikian, Garabed
• Format: Journal Article
• Language: English
• Published: 01.09.1999
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.1432-1327.1999.00640.x

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 Ca 2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca 2+ ions with a K d of 0.080 ± 0.010 µ m 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 Δ G H 2 O D 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 Ca 2+ .