Related Experiment Videos
Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and
L S Kraakman1, J Winderickx, J M Thevelein
1Laboratorium voor Moleculaire Celbiologie, Instituut voor Plantkunde en Microbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium.
The Biochemical Journal
|September 24, 1999
Summary
Baker's yeast hexokinases Hxk1 and Hxk2 regulate sugar repression and Ras-cAMP signaling. Catabolite repression establishment depends on hexokinase's phosphoryl transfer reaction, not just sugar binding.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- Hexokinases PI (Hxk1) and PII (Hxk2) in Saccharomyces cerevisiae are crucial for Ras-cAMP pathway activation and catabolite repression.
- Hxk2 is essential for glucose repression, while Hxk1 or Hxk2 can maintain fructose repression.
- Previous research suggested a link between hexokinase catalytic activity and glucose repression, but glucose 6-phosphate was not identified as the direct trigger.
Purpose of the Study:
- To investigate the structural requirements of Hxk2 for sugar-induced signaling using site-directed mutagenesis.
- To elucidate the roles of specific amino acids in hexokinase binding, catalysis, and conformational changes related to signaling.
Main Methods:
- Site-directed mutagenesis of key amino acids in Hxk2, including those involved in ATP binding, phosphoryl transfer, and substrate cleft closure.
- Assessing the effects of these mutations on hexokinase catalytic activity, sugar binding affinity, Ras-cAMP pathway activation, and catabolite repression in vivo.
- Utilizing 2-deoxyglucose to further probe the relationship between catalysis and repression.
Main Results:
- The ATP-binding Lys-111 is not essential for Hxk2's in vivo catalysis or signal triggering.
- Substitution of catalytic-center Asp-211 abolished catalytic activity but retained high-affinity sugar binding, yet failed to induce cAMP activation or catabolite repression.
- Mutation of Ser-158 affected glucose-induced repression but not fructose-induced repression; 2-deoxyglucose sustained repression despite low catalytic activity.
Conclusions:
- Catabolite repression establishment relies on the hexokinase phosphoryl transfer reaction, likely involving a transition intermediate and conformational changes.
- Hxk2's role in Ras-cAMP activation appears directly linked to its catalytic function.
- These findings propose a model differentiating the mechanisms of hexokinase involvement in catabolite repression versus Ras-cAMP pathway activation.