Active-site serine phosphate and histidine residues of phosphoglucomutase: pH titration studies monitored by 1H and 31P NMR spectroscopy.

1H and 31P NMR pH titrations were conducted to monitor changes in the environment and protonation state of the histidine residues and phosphoserine group of rabbit muscle phosphoglucomutase on binding of metal ions at the activating site and of substrate (glucose phosphate) at the catalytic site. Imidazole C epsilon-H signals from 8 of the 10 histidines present in the free enzyme were observed in 1H NMR spectra obtained by a spin-echo pulse sequence at 470 MHz; their pH (uncorrected pH meter reading of a 2H2O solution measured with a glass electrode standardized with H2O buffer) titration properties (in 99% 2H2O) were determined. Three of these histidine residues, which have pKa values ranging from 6.5 to 7.9, exhibited an atypical pH-dependent perturbation of their chemical shifts with a pHmid of 5.8 and a Hill coefficient of about 2. Since none of the observed histidines has a pKa near 5.8, it appears that these three histidines interact with a cluster consisting of two or more groups which become protonated cooperatively at this pH. Binding of Cd2+ at the activating site of the enzyme abolishes the pH-dependent transition of these histidines; hence, the putative anion cluster may constitute the metal ion binding site, or part of it. Two separate 31P NMR peaks from phosphoserine-116 of the phosphoenzyme were observed between pH 6 and 9. Apparently, the metal-free enzyme exists as a pH-dependent mixture of conformers that provide two different environments, I and II, for the enzymic phosphate group; the transition of the phosphate group between these two environments is slow on the NMR time scale.(ABSTRACT TRUNCATED AT 250 WORDS)