Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression

In baker's yeast (Saccharomyces cerevisiae) the hexokinases PI (Hxk1) and P II (Hxk2) are required for triggering of the activation of the Ras-cAMP pat hway and catabolite repression. Specifically, Hxk2 is essential for the est ablishment of glucose repression, whereas either Hxk1 or Hxk2 can sustain f ructose repression. Previous studies have suggested that the extent of gluc ose repression is inversely correlated with hexokinase catalytic activity a nd hence with an adequate elevation of intracellular sugar phosphate levels . However, several lines of evidence indicate that glucose 6-phosphate is n ot the trigger of catabolite repression in yeast. In the present study we e mployed site-directed mutagenesis of amino acids important for the binding of sugar and ATP, for efficient phosphoryl transfer and for the closure of the substrate-binding cleft, to obtain an insight into the structural requi rements of Hxk2 for sugar-induced signalling. We show that the ATP-binding Lys-111 is not essential for catalysis in vivo or for signal triggering. Su bstitution of the catalytic-centre Asp-211 caused loss of catalytic activit y, but high-affinity sugar binding was retained. However, this was not suff icient to cause cAMP activation nor catabolite repression. Mutation of Ser- 158 abrogated glucose-induced, but not fructose-induced, repression. Moreov er, 2-deoxyglucose sustained repression despite an extremely low catalytic activity. We conclude that the establishment of catabolite repression is de pendent on the onset of the phosphoryl transfer reaction on hexokinase and is probably related to the stable formation of a transition intermediate an d concomitant conformational changes within the enzyme. In contrast, the ro le of Hxk2 in Ras-cAMP activation seems to be directly connected to its cat alytic function. The implications of this model are discussed.