Structural stability of E. coli transketolase to temperature and pH denaturation

  • Jahromi R
  • Morris P
  • Martinez-Torres R
 et al. 
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We have previously shown that the denaturation of TK with urea follows a non-aggregating though irreversible denaturation pathway in which the cofactor binding appears to become altered but without dissociating, then followed at higher urea by partial denaturation of the homodimer prior to any further unfolding or dissociation of the two monomers. Urea is not typically present during biocatalysis, whereas access to TK enzymes that retain activity at increased temperature and extreme pH would be useful for operation under conditions that increase substrate and product stability or solubility. To provide further insight into the underlying causes of its deactivation in process conditions, we have characterised the effects of temperature and pH on the structure, stability, aggregation and activity of Escherichia coli transketolase. The activity of TK was initially found to progressively improve after pre-incubation at increasing temperatures. Loss of activity at higher temperature and low pH resulted primarily from protein denaturation and subsequent irreversible aggregation. By contrast, high pH resulted in the formation of a native-like state that was only partially inactive. The apo-TK enzyme structure content also increased at pH 9 to converge on that of the holo-TK. While cofactor dissociation was previously proposed for high pH deactivation, the observed structural changes in apo-TK but not holo-TK indicate a more complex mechanism. © 2011 Elsevier B.V.

Author-supplied keywords

  • Biocatalysis
  • Denaturation
  • Stability
  • Transketolase
  • Tryptophan fluorescence

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  • Paul DalbyUniversity College London

  • Raha R F Jahromi

  • Phattaraporn Morris

  • Ruben J. Martinez-Torres

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