A MUTATION SER(213) ASN IN THE HEXOKINASE-1 FROM SCHIZOSACCHAROMYCES-POMBE INCREASES ITS AFFINITY FOR GLUCOSE/

Alignment of amino acids of the region implicated in glucose binding f rom a series of hexokinases showed that Schizosaccharomyces pombe hexo kinase 1 had a Ser residue in a place where all other kinases had an A sn. We changed an AGT codon to AAT to place an Asn in the Ser(213) pos ition. This mutation decreased K-m for glucose from 9.4 mM to 1.6 mM a nd the ratio V-max (Fructose)/V-max (Glucose) from 5 to 2.5, Also the K-m for 2-deoxyglucose decreased from 2.7 mM to 0.8 mM, A mutation in the similar position of S. pombe hexokinase 2 (Asn(196)/Ser) increased the K-m for glucose from 0.16 mM to 0.56 mM, Fermentation of glucose is not detectable in a S. pombe mutant with only hexokinase 1 activity but expression of the hxk1(S213/N) gene conferred ability to ferment the sugar. While the mutated hexokinase 1 partially mimicked S. cerevi siae hexokinase II in catabolite repression of invertase, the wild typ e one could not substitute for it. (C) 1998 Academic Press.