Yeast 6-phosphofructo-2-kinase: sequence and mutant.
We have reported yeast 6-phosphofructo-2-kinase (EC 2.7.1.105) as having a ca. 96-kDa subunit size, as well as isolation of its structural gene, PFK26. Sequencing now shows an open reading frame of 827 amino acids and 93.5 kDa. The deduced amino acid sequence has 42% identity with the 55-kDa subunit of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver with extra material at both ends. Although the yeast sequence is especially similar to the liver one in its bisphosphatase domain, the essential His-258 of the liver enzyme is, in yeast, a serine, which may explain the apparent lack of bisphosphatase activity. Also, the yeast enzyme known to be activated via protein kinase A, has a putative phosphorylation site near its C-terminus and lacks the N-terminal phosphorylation sequence involved in inhibition of the liver enzyme. In a chromosomal null mutant strain, pfk26::LEU2, activity was marginal and the protein was not detectable as antigen. The mutant strain grew well on glucose and contained a near-normal level of fructose 2,6-P2. But in its growth on pyruvate, by contrast with the wild-type strain, no fructose 2,6-P2 was detectable, and it did not form after glucose addition in the presence of cycloheximide either. Such resting cells, however, metabolized glucose at the normal high rate. Glucose addition to the pfk26 mutant strain in the absence of cycloheximide, on the other hand, caused a ca. 10% normal rate of fructose 2,6-P2 accumulation, presumably employing a glucose-inducible second enzyme. Using strains also lacking 6-phosphofructo-1-kinase, affinity chromatography revealed the second enzyme as a minor peak amounting to 6% of 6-phosphofructo-2-kinase activity in a PFK26 strain and as the sole peak, in similar amount, in a pfk26 mutant strain.
Kretschmer
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Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003513:Cycloheximide / Q000494:pharmacology; D004271:DNA, Fungal / Q000737:chemistry; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D025481:Phosphofructokinase-2; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D003513:Cycloheximide; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D005947:Glucose