Open AccessThe effect of Glu75 of staphylococcal nuclease on enzyme activity, protein stability and protein unfolding
Author(s) -
Chen Hueih Min,
Dimagno Theodore J.,
Wang Wei,
Leung Eric,
Lee ChengHao,
Chan Sunney I.
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00317.x
Subject(s) - mutant , wild type , denaturation (fissile materials) , mutant protein , chemistry , enzyme , enzyme kinetics , protein folding , nuclease , isothermal microcalorimetry , enthalpy , microbiology and biotechnology , biochemistry , biology , active site , nuclear chemistry , physics , quantum mechanics , gene
Staphylococcal nuclease mutants, E57G and E75G, were generated. A comparison of the kinetic parameters both for mutants and wild‐type protein shows that the Michaelis constants ( K m ) were almost identical for the wild‐type protein and E57G mutant. An approximately 30‐fold decrease in K m compared with the wild‐type protein was observed for the E75G mutant. The turnover numbers for the enzyme ( k cat ) were higher with both the wild‐type protein and the E57G mutant (3.88 ± 0.21 × 10 3 s −1 and 3.71 ± 0.28 × 10 3 s −1 ) than with the E75G mutant (3.04 ± 0.02 × 10 2 s −1 ). The results of thermal denaturation with differential scanning microcalorimetry indicate that the excess calorimetric enthalpy of denaturations, Δ H cal , was almost identical for the wild‐type protein and E57G mutant (84.1 ± 6.2 kcal·mol −1 and 79.3 ± 7.1 kcal·mol −1 , respectively). An approximately twofold decrease in Δ H cal compared with the wild‐type protein was observed for the E75G mutant (42.7 ± 5.5 kcal·mol −1 ). These outcomes imply that Glu at position 75 plays a significant role in maintaining enzyme activity and protein stability. Further study of the unfolding of the wild‐type protein and E75G mutant was conducted by using time‐resolved fluorescence with a picosecond laser pulse. Two fluorescent lifetimes were found in the subnanosecond time range. The faster lifetime ( τ 2 ) did not generally vary with either pH or the concentration of guanidinium hydrochloride (GdmHCl) in the wild‐type protein and the E75G mutant. The slow lifetime ( τ 1 ), however, did vary with these parameters and was faster as the protein is unfolded by either pH or GdmHCl denaturation. The midpoints of the transition for τ 1 are pH 3.5 and 5.8 for the wild‐type protein and E75G mutant, respectively, and the GdmHCl concentrations are 1.1 m and 0.6 m for the wild‐type protein and E75G mutant, respectively. Parallel steady‐state fluorescence measurements have also been carried out and the results are in general agreement with the time‐resolved fluorescence experiments, indicating that Glu at position 75 plays an important role in protein unfolding.