Characterization of the histidine residues in alkaline phosphatase by carbon-13 nuclear magnetic resonance.

Abstract

beta,beta-[gamma-13C]Dideuteriohistidine has been biosynthetically incorporated into alkaline phosphatase from Escherichia coli and utilized as a nonperturbing 13C nuclear magnetic resonance (NMR) probe of the environments of the histidine residues in this zinc metalloenzyme. The 13C NMR spectrum of the labeled enzyme exhibits 9 separate resonances arising from the 10 histidine residues located in each of the symmetrically disposed subunits of the dimer. The excellent resolution and large chemical shift range (14 ppm) displayed by these signals are direct consequences of the sensitivity of the histidine gamma-carbon chemical shift to the ionization state and tautomeric form of the imidazole side chains and the coordination of several of these to metal ion. The environments of the individual histidine residues were inferred by investigating the chemical shift responses of their 13C resonances to enzyme metal composition, pH, and inhibitor binding. Additional information concerning their motional freedom was obtained from spin relaxation measurements which were analyzed in terms of the contributions expected from intramolecular 13C-1H and 13C-14N dipolar relaxation and chemical shift anisotropy. The combined results indicate that 4 of the 10 histidines, the only ones that titrate with pH, are surface residues located relatively remote from the active site. Of the six nontitrating residues, one appears to be buried in a solvent-inaccessible region of the protein. Three others are almost certainly involved in metal ion ligation to active-site metal ion(s), two via their N pi nitrogen atoms and the other via N pi. The spectral characteristics of the remaining two histidine residues strongly suggest they are also located at or near the active site. One or both may also participate in metal ion coordination, although the current evidence for this is inconclusive.