Kinetic, binding, and NMR studies of perdeuterated yeast phosphoglycerate kinase and its interactions with substrates.
Perdeuterated yeast phosphoglycerate kinase (2HPGK) was prepared from yeast cells grown in 99.9% 2H2O and an acid hydrolysate from deuterated algal cells. Kinetic and binding studies suggested that perdeuterated enzyme was similar to the isotopically normal PGK. The use of 2HPGK not only eliminated the spectral overlap between the enzyme and substrate nuclear Overhauser effect (NOE) cross-peaks, but also permitted observation of weak transfer NOE cross-peaks between the substrate protons that are greater than 4 A apart. Intensity of NOE cross-peaks was used to determine the interproton distances of enzyme-bound Mg(II)dATP. These distances were then used in model building studies to determine the conformation of Mg(II)dATP. The average conformation of enzyme-bound dATP is anti with O4'/C2' endo ribose pucker and trans, gauche about the C4'-C5' bond. Although many spin diffusion pathways were eliminated by protein deuteration, spin diffusion was still observed between the protons of the substrate at mixing times longer than 25 ms.
Shibata
C G
CG
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840, USA.
Gregory
J D
JD
Gerhardt
B S
BS
Serpersu
E H
EH
D001665:Binding Sites; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D003903:Deuterium; D005988:Glyceric Acids / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D003838:Deoxyadenine Nucleotides; D005988:Glyceric Acids; C005156:3-phosphoglycerate; D003903:Deuterium; D010735:Phosphoglycerate Kinase; C026600:2'-deoxyadenosine triphosphate