Mechanistic studies of yeast phosphoglycerate kinase using isotopically-labeled enzyme analogues and NMR spectroscopy

Author

Kameshwari Mantha Pappu

Date of Award

12-1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin H. Serpersu

Committee Members

Cynthia B. Peterson, Elizabeth E. Howell, Solon Georghiou

Abstract

Perdeuterated yeast phosphoglycerate kinase ([²H]PGK) with only histidine side chains of normal [¹H] isotopic composition was prepared biosynthetically. All the imidazole C2 and C4 proton resonances of the eight histidine residues of the isotope-hybrid enzyme ([¹H]His [²H]PGK) were detected by ¹H nuclear magnetic resonance (NMR) spectroscopy, which permitted detailed structure and function studies. pH titrations of isotope-hybrid PGK revealed that most of the imidazole C2 and C4 protons of the histidine residues titrate within the pH range of 5 to 9. Titration of isotope-hybrid PGK with substrates showed that the formation of binary enzyme•MgdATP and ternary enzyme•MgdATP•Glycerol-3-phosphate (G3P) complexes caused slight shifts and broadening in some of the histidine C2 and C4 proton resonances. Also, proton signals of bound substrates were visible without spectral overlap and broadening in the ¹H NMR spectrum even with a substrate-to-enzyme ratio of less than 1:2 (mol/mol). Although the protein concentration was very high (1.5 mM), no intraprotein cross peaks other than those of intraresidue histidine NOE cross peaks were observed in a two-dimensional NOESY spectrum of enzyme•MgdATP•G3P complex. In addition, intrasubstrate NOE cross peaks were clearly visible with a substrate-to-enzyme ratio of 1.5:1 (mol/mol). Intermolecular NOE cross peaks between the protons of G3P and histidine C2 proton resonances were also observed, suggesting the proximity of bound triosephosphate and two histidine residues. The paramagnetic effect of a substrate analogue CrATP on some of the histidine C2 and C4 proton resonances indicated that the formation of a ternary complex caused significant conformational changes in the enzyme. Thus, labeling of a perdeuterated protein with a specific protiated amino acid permits detailed ¹H NMR studies on the structure and function of large enzymes, as demonstrated with yeast PGK. Exchange-inert RhATP complexes were used to obtain structural information about the catalytically active form of yeast PGK. β, γ-bidentate RhATP showed a single turnover substrate activity with PGK. Transfer of the phosphoryl group between ATP and 3-phospho-D-glycerate (3PGA) occurred with both substrates in the coordination sphere of the metal ion. Because of the slow ligand exchange rate of Rh(III), the reaction product 1,3- bisphosphoglycerate (1,3-dPGA) remained coordinated to the metal ion. During the course of the reaction the enzyme was inactivated due to the coordination of at least one protein side chain to Rh(III). Thus the product RhADP•1,3-dPGA remained bound to the enzyme even after removal of excess substrates. The results suggest that the metal ion may not only act as an electron sink to activate the electrophile, but may also align the substrates optimally for phosphoryl transfer by coordination to both substrates. Therefore, entry of 3PGA into the coordination sphere of metal of a metal- ATP complex may start the proposed hinge-bending motion of yeast PGK to form a "closed" active site between the two substrate binding domains of the enzyme. Suicide substrate β, γ-bidentate RhATP was also used to map the metal ion binding site in yeast PGK. Cleavage of the RhATP-inactivated enzyme with pepsin and subsequent separation of peptides by reverse-phase HPLC 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 5. Of the four glutamates present in the C-terminal peptide, Glu 398 may be a likely metal coordination site. Therefore, the C-terminal peptide is important in PGK catalysis, in part, since it facilitates the coordination of metal ion of the metal-ATP substrate. This may then cause the C-terminal peptide to extend towards the N-terminal domain and form the "closed" active site. Results suggest that one or more side chains of the enzyme may be coordinated to the metal ion in the PGK•3PGA•RhATP complex. Therefore, exchange-inert metal-ATP analogues may be used to determine metal coordination sites on kinases and other metal-ATP utilizing enzymes.

Recommended Citation

Pappu, Kameshwari Mantha, "Mechanistic studies of yeast phosphoglycerate kinase using isotopically-labeled enzyme analogues and NMR spectroscopy. " PhD diss., University of Tennessee, 1996.
https://trace.tennessee.edu/utk_graddiss/9825