Proton nuclear magnetic resonance (NMR) spectroscopy has been used to
investigate the interaction of yeast phosphoglycerate kinase (PGK) with its
phosphate containing substrates, ATP and 3-phosphoglycerate (3-PG). The
application of one-dimensional and, for the first time, two-dimensional proton
NMR techniques to this large protein has enabled specific resonance
assignments to be made. Assignment has been aided by the investigation of
specifically deuterated protein and site-specific mutant forms of the protein,
including the isolated N- and C-domains.
The effects of ATP and 3-PG binding on the proton NMR spectrum of yeast
PGK have been characterised and the assigned resonances used as local probes
of structural and dynamic changes. Two binding sites have been determined
for the nucleotide substrate, ATP, the occupancies of which are dependent on
Mg2+ concentration. One site corresponds to the catalytic site determined
crystallographically. A single binding site was found for 3-PG. This binding
was shown to cause highly specific conformational changes throughout the
N-domain and the interdomain region, which involve the relative movement of
at least three α-helices. Investigation of 3-PG binding to several
site-specific mutant forms of yeast PGK revealed a critical role for arginine
168 in the propagation of these changes.
The general binding of anions to yeast PGK was investigated using the
paramagnetic probes [Cr(CN)6]3- and [Fe(CN)6]3-, and the diamagnetic anion
[Co(CN)6]3-. The primary anion binding site was determined from [Cr(CN)6]3-
broadening data and found to share some side-chains involved in 3-PG
binding, namely histidine 62 and arginine 168. Evidence for a secondary anion
site was found. The anion binding data is discussed in view of the complex
activation/inhibition effects of anions on the catalytic activity.
Investigation of the isolated N- and C-domains showed that both can fold
independently and confirmed that the C-domain is a nucleotide binding
domain. It appears that the presence of the interdomain residues and/or the
C-terminal peptide are necessary for 3-PG binding to the N-domain.
This work shows that the specificity of the substrates is in binding, as
expected, but also in the motions induced in the protein as a whole.</p