A New Metal-Binding Site for Yeast Phosphoglycerate Kinase as Determined by the Use of a Metal-ATP Analog

Abstract

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.

Use of Viscogens, dNTPαS, and Rhodium(III) as Probes in Stopped-Flow Experiments To Obtain New Evidence for the Mechanism of Catalysis by DNA Polymerase β,

“…nucleotide complexes have been reported for Cr(III), Co(III) (41), and Rh(III) (24). Of these, Rh(III) has been used least frequently in enzymatic studies (24,(42)(43)(44)(45), and has never been used to study polymerases. Of the three metal nucleotide complexes studied, Rh(III)-NTP has been shown to be structurally most similar to Mg-(II)NTP.…”

Substrate-Assisted Movement of the Catalytic Lys 215 during Domain Closure: Site-Directed Mutagenesis Studies of Human 3-Phosphoglycerate Kinase

“…PGK is a typical two-domain hinge-bending enzyme (16), with a highly conserved structure (17,18). The N-terminal domain has a basic patch region for binding of 3-PG (19)(20)(21)(22)(23)(24)(25)(26)(27), and the C-terminal domain contains the binding site for the nucleotide substrates, MgADP and MgATP (20)(21)(22)(23)(27)(28)(29)(30)(31)(32)(33)(34), as revealed by both crystallographic (19)(20)(21)(22)(23)(24)(28)(29)(30) and NMR chemical shift perturbation (25,27,(31)(32)(33)(34) as well as relaxation (25,27,(31)(32)(33)(34) studies. No such definitive information is available for the interaction with the highly unstable 1,3-BPG.…”