A new metal-binding site for yeast phosphoglycerate kinase as determined by the use of a metal-ATP analog.
Suicide substrate beta, gamma-bidentate Rh(III)ATP (RhATP) was used to map the metal ion-binding site in yeast phosphoglycerate kinase (PGK). Cleavage of the RhATP-inactivated enzyme with pepsin and subsequent separation of peptides by reverse-phase high-performance liquid chromatography gave two Rh-nucleotide bound peptides. One of the peptides corresponded to the C-terminal residues of PGK, and the other to a part of helix V. Of the four glutamates present in the C-terminal peptide, Glu 398 may be a likely metal coordination site. Therefore, importance of the C-terminal residues in PGK catalysis may be attributed, in part to the coordination of metal ion of the metal-ATP substrate. Metal coordination may then align the C-terminal peptide to extend toward the N-terminal domain and form the "closed" active site. Results presented in this paper suggest that one or more side chains of the enzyme may be coordinated to the metal ion in the PGK.3-phospho-D-glycerate-RhATP complex, and that exchange-inert metal-ATP analogs could be used to determine metal coordination sites on kinases and other metal-ATP-utilizing enzymes.
Pappu
K M
KM
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville 37996-0840, USA.
Kunnumal
B
B
Serpersu
E H
EH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005658:Fungi / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012238:Rhodium / Q000378:metabolism*; D013053:Spectrophotometry
D004791:Enzyme Inhibitors; D008670:Metals; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D012238:Rhodium; D010735:Phosphoglycerate Kinase; D010434:Pepsin A