Phosphorus-31 nuclear magnetic resonance pH titration studies of the phosphoproteins tropomyosin and glycogen phosphorylase a

Abstract

31P nuclear magnetic resonance (NMR), pH titration studies of the phosphoproteins tropomyosin and glycogen phosphorylase a (in the presence of the inhibitor glucose) show that the resonances for the phosphoserine regulatory sites shift with pH. Analysis of line widths indicates that both residues have considerable mobility. These results are in agreement with studies on similar phosphorylated sites on other proteins, leading us to propose that mobility is a general feature of such regulatory sites. pH titration studies on a series of model compounds have indicated that an empirical correlation exists between the Hill coefficient n (a measure of the cooperativity of the titration curve) and the presence of charged groups in the vicinity of the phosphoryl moiety. Moreover, these studies showed that within one class of similarly substituted phosphorus compounds the chemical shifts, the titration behaviour, and the pKa2 were comparable and allow for easy identification of these compounds. The pKa2 values for phosphoserine residues of proteins are in general slightly higher than those of phosphomonoester-containing small compounds. The titration data prompt our estimation that the maximal amount of energy associated with a salt linkage between a phosphoseryl side chain and a positively charged group is about 5 kcal (1 cal = 4.1868 J).

Phosphorus-31 nuclear magnetic resonance study of the active site phosphohistidine and regulatory phosphoserine residues of rat liver ATP-citrate lyase

“…This enzyme, with its two classes of phosphorylated groups, provides further confirmation for suggestions (Vogel & Bridger, 1982a,b;Vogel et al, 1982;Vogel, 1984) that the structural or regulatory phosphate groups would generally be found on the surface of enzymes in environments where their mobility would be unrestricted, while phosphate groups that served as catalytic intermediates would be sequestered in buried environments within the active site, with essentially no residual mobility and with greatly reduced access to solvent. Moreover, these results confirm the site of phosphorylation at the active center as a histidine residue, inconsistent with previous suggestions (Inoue et al, 1966b) of the presence of acyl phosphate.…”

“…Using the calculations and theory outlined by Vogel et al (1982), developed for the enzyme-bound and relatively im- mobile 3-phosphohistidine residue of E . coli succinyl-CoA synthetase, we have estimated the line width expected for 3-phosphohistidine bound to an enzyme of molecular weight 440000.…”

Study of the phosphorylatable light chains of skeletal and gizzard myosins by nuclear magnetic resonance spectroscopy

“…The titration end-points were determined by extrapolation to high pH and by reference to the overall shift upon ionization (4+0.5p.p.m.) found in previous studies of monoester phosphates (Brauer & Sykes, 1984;Vogel, 1983;Vogel & Bridges, 1983). from threonine yCH, groups also indicates that segments of the structure encompassing the remaining threonine residues of the molecule 50,78,82,98,111,122, 123 and 158) possess differentially restricted mobility compared to the N-terminal region. The conclusion that these other regions of the structure of the skeletal muscle light chain are relatively more structured is supported by the observed 'H n.m.r.…”

“…data for serine and threonine phosphates previously reported in proteins (e.g. Vogel & Bridges, 1983;Brauer & Sykes, 1984). Precipitation of the proteins below pH 6 precluded direct or similar determination of the chemical shift position associated with the fully protonated state of each protein phosphate group.…”

“…To obtain a numerical evaluation of apparent pKa values rather than a qualitative comparison with the parameters found for the free monoester phosphates, chemical shift values for the fully protonated species were inferred from the data for serine phosphates (overall ionization induced shift of 4 + 0.5 p.p.m. ; Vogel & Bridges, 1983;Brauer & Sykes, 1984). The least-square fit of the titration data using these end-point values is reported.…”