A mutation Ser213/Asn in the hexokinase 1 from Schizosaccharomyces pombe increases its affinity for glucose.
Alignment of amino acids of the region implicated in glucose binding from a series of hexokinases showed that Schizosaccharomyces pombe hexokinase 1 had a Ser residue in a place where all other kinases had an Asn. We changed an AGT codon to AAT to place an Asn in the Ser213 position. This mutation decreased Km for glucose from 9.4 mM to 1.6 mM and the ratio Vmax (Fructose)/Vmax (Glucose) from 5 to 2.5. Also the Km for 2-deoxyglucose decreased from 2.7 mM to 0.8 mM. A mutation in the similar position of S. pombe hexokinase 2 (Asn196/Ser) increased the Km 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. cerevisiae hexokinase II in catabolite repression of invertase, the wild type one could not substitute for it.
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Instituto de Investigaciones Biomédicas Alberto Sols, C.S.I.C., Unidad de Bioquímica y Genética de Levaduras, Madrid, 28029, Spain.
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Gancedo
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D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D005285:Fermentation; D005947:Glucose / Q000378:metabolism*; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010101:Oxygen Consumption; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics
D007527:Isoenzymes; D011994:Recombinant Proteins; D012694:Serine; D001216:Asparagine; D006593:Hexokinase; D005947:Glucose