The unfolding of shikimate kinase (SK) from Erwinia chrysanthemi by urea and its subsequent refolding on dilution of the denaturing agent has been studied in detail [Eur. J. Biochem. 269 (2002) 2124]. Comparison of the effects of urea on the enzyme with those of guanidinium chloride (GdmCl) and NaCl indicated that chloride ions significantly weakened the binding of shikimate. This finding prompted a more detailed examination of the effects of salts on the structure, function and stability of the enzyme; the effects of NaCl and Na(2)SO(4) were investigated in detail. These salts have very small effects on the secondary structure as judged by far UV CD circular dichroism (CD), although the near UV CD spectra suggest that some limited changes in the environment of aromatic amino acids may occur. Both salts inhibit SK activity and analysis of the kinetic and substrate binding parameters point to a complex mechanism for the inhibition. Inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea. When the enzyme is unfolded by incubation in 4 M urea, addition of NaCl or Na(2)SO(4) leads to a relatively slow refolding of the enzyme as judged by the regain of native-like CD and fluorescence. In addition, the refolded enzyme can bind shikimate, though more weakly than the native enzyme. However, the refolded enzyme does not appear to be capable of binding nucleotides, nor does it possess detectable catalytic activity. The refolding process brought about by addition of salt in the presence of 4 M urea is not associated with any change in the fluorescence of the probe 8-anilino-1-naphthalenesulfonic acid (ANS), indicating that an intermediate formed by hydrophobic collapse is unlikely to be significantly populated. The results point to both specific and general effects of salts on SK. These are discussed in the light of the structural information available on the enzyme. Study holds ProTherm entries: 14310, 14311, 14355, 14356, 14357, 14358, 14359, 14360 Extra Details: 0.4 mM dithiothreito (DTT) was addes as additive. Shikimate kinase; Protein stability; Salt effect; Protein unfolding; Protein refolding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||8|
|Proteins||Shikimate kinase 2 ; Shikimate kinase 2|
|Assays/Quantities/Protocols||Experimental Assay: Cm ionic:KCl,MgCl2: 5 mM,2.5 mM, buffers:Tris-HCl: 35 mM ; Experimental Assay: Cm buffers:MOPS: 50 mM, ionic:K2SO4,MgSO4: 2.5 mM,2.5 mM ; Experimental Assay: Cm buffers:Tri-HCl: 35 mM, ionic::|
|Libraries||Mutations for sequence MTEPIFMVGARGCGKTTVGRELARALGYEFVDTDIFMQHTSGMTVADVVAAEGWPGFRRRESEALQAVATPNRVVATGGGMVLLEQNRQFMRAHGTVVYLFAPAEELALRLQASPQAHQRPTLTGRPIAEEMEAVLREREALYQDVAHYVVDATQPPAAIVCELMQTMRLPAA|