Proton Magnetic Resonance Studies of the States of Ionization of Histidines in Native and Modified Subtilisins†

Guillermo Tous, Laszlo Polgar, Frank Jordan

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A technique was developed to exchange the backbone -N-H protons in D2O in the native subtilisins Carlsberg and BPN (Novo) that resulted in clearly resolved proton resonances in the aromatic region of the nuclear magnetic resonance spectrum. pH titration curves for four of the five histidine C2-H resonances in subtilisin Carlsberg and five of the six in subtilisin BPN between 7.5 and 8.8 ppm downfield from 4,4-dimethyl-4-silapentane-l-sulfonic acid sodium salt provided microscopic pka's between 6.3 and 7.2 for both sources of the enzyme at ambient (~22 °C) probe temperature. A resonance that titrated with a pkapp of 7.35 ± 0.05 was observed in the 1H spectra only of the diisopropylphosphoryl derivatives of the subtilisins from both sources. The 31P NMR pH titration of the same preparations under identical conditions of solvent (D2O) and temperature gave a pKapp = 7.40 ± 0.05 of the single titratable resonance. Both observations must pertain to His-64 at the active center. A resonance smaller than the others and titrating with a pKapp of 7.2 could also be observed in the native enzymes. This resonance was assigned to the catalytic center histidine since its pK corresponded to that derived from kinetic studies. No major perturbations in the chemical shifts or the pKs derived from the pH dependence of the observed resonances were apparent in the presence of saturating concentrations of the two putative transition-state analogues phenylboronic acid and bis[3,5-(trifluoromethyl)phenyl]boronic acid and in monoisopropylphosphorylsubtilisin. It can be concluded that the C2-H resonance corresponding to His-64 in native subtilisins is difficult to observe perhaps on account of the limited mobility of this side chain compared to its mobility in the diisopropylphosphoryl derivative.

Original languageEnglish
Pages (from-to)7711-7717
Number of pages7
Issue number26
Publication statusPublished - dec. 1 1985


ASJC Scopus subject areas

  • Biochemistry

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