Cloning and expression of Saccharomyces cerevisiae inorganic pyrophosphatase

Lee Franklin Kolakowski, University of Pennsylvania


S. cerevisiae inorganic pyrophosphatase (PPase) (E.C. is among the best characterized simple phosphoryl-transfer enzymes. PPase's activity is the cleavage of pyrophosphate (PP$\sb{\rm i}$), a by-product of many different biological processes, including charging of amino acids and nucleic acid synthesis. The gene for PPase (PPA1) has been cloned by hybridization of oligonucleotide probes, based on the known amino acid sequence (Cohen, S. A., Sterner, R., Keim, P. S., & Heinrikson R. L. 1978 J. Biol. Chem. 253, 3, 889-897) with a genomic S. cerevisiae library. The 1612 basepair nucleotide sequence from the clone gives a deduced 286 amino acid sequence that differs in nine positions from that previously published. The PPA1 gene appears to be a single copy gene located on the left arm of chromosome II next to the Copy-I locus of histones H3 and H4. Alignments of the amino acid sequences of PPases have been examined in light of crystallographic and chemical modification results placing specific amino acids at the active site of the yeast enzyme. The major results of these analyses are: (1) PPases across evolution share a common catalytic core of 15 amino acids, and (2) PPases exist in two major quaternary structures. PPase thus, appears to be an example of enzymes from widely divergent species that conserve common functional elements in context of overall sequence variation. Using an inducible promoter in the expression vector pKK233-2 and synthetic linkers, and the PPA1 gene, a plasmid (pFK-E45) was constructed which expresses yeast PPase at high levels in E. coli. PPase produced under this system appears to fold properly and retain wild type catalytic activity. The construction of a heterologous expression system for the PPA1 gene in E. coli will allow rapid testing of enzymatically important residues.

Subject Area

Biochemistry|Molecular biology

Recommended Citation

Kolakowski, Lee Franklin, "Cloning and expression of Saccharomyces cerevisiae inorganic pyrophosphatase" (1992). Dissertations available from ProQuest. AAI9227702.