Guanylate kinase from Saccharomyces cerevisiae. Isolation and characterization, crystallization and preliminary X-ray analysis, amino acid sequence and comparison with adenylate kinases.
This paper describes a large-scale purification of guanylate kinase (ATP + GMP in equilibrium ADP + GDP) from Saccharomyces cerevisiae, the crystallization of the enzyme and preliminary X-ray investigations. Furthermore the complete amino acid sequence of the enzyme has been determined and was compared to adenylate kinase sequences. 1. Guanylate kinase was purified in five steps to homogeneity: crude extract, ion-exchange chromatography, affinity chromatography and gel filtration twice. 2. The enzyme was crystallized to single octahedral bipyramids with sizes up to 500 x 200 x 150 microns 3. Preliminary X-ray results are given. 3. The final sequence shows 186 amino acids (Mr = 20,548), containing one cysteine and one tryptophan. It was determined from peptides of five cleavages of the whole protein. Three cleavages were used for determination of the whole polypeptide chain. From the other two, only some peptides were used to secure overlaps and the cysteine position. The N-terminal blocking group was identified by 1H-NMR spectroscopy. 4. Since guanylate kinase shows the mononucleotide binding pattern GXXGXGK, it was compared to other proteins containing this pattern. But no further homology signal could be detected. A comparison with adenylate kinases revealed significant similarity in another chain segment. This led to the conclusion that guanylate kinase is at least partially homologous to the adenylate kinases.
Berger
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Institut für Organische Chemie und Biochemie der Universität Freiburg im Breisgau.
Schiltz
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Schulz
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D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D051528:Guanylate Kinases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D010770:Phosphotransferases / Q000302:isolation & purification*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D014961:X-Ray Diffraction
D010446:Peptide Fragments; D010770:Phosphotransferases; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases; D010447:Peptide Hydrolases; D003488:Cyanogen Bromide