High-Resolution Nitrogen-15 Nuclear Magnetic Resonance Studies of α-Lytic Protease in Solid State. Direct Comparison of Enzyme Structure in Solution and in the Solid State

Tai Huang Huang, William W. Bachovchin, Robert G. Griffin, Christopher M. Dobson

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Histidine enriched in N in the imidazole nitrogens was incorporated into the catalytic triad of α-lytic protease, and high-resolution solid-state 15N NMR spectra of lyophilized enzyme powders were recorded. The lyophilized powders were prepared from aqueous solutions with pH values ranging from 4.9 to 9.3. The behavior of the 15N resonances as a function of “pH” in these solid samples closely parallels that observed previously in the corresponding solution-state study, with the exception that in the powders proton exchange at His-57 is slow on the NMR time scale whereas in solutions it is fast. Thus, the 15N isotropic shifts demonstrate that the Nπ-H tautomer of His-57 predominates in powders prepared at high pH and that Nπ(H) participates in a strong hydrogen bond, as the hydrogen-bond donor, in powders prepared at both high pH and low pH. The simplest interpretation of these results is that the active site catalytic triad structure of Asp-His-Ser is maintained in these lyophilized powders. Because Asp-102 and His-57 are sequentially separated, their interaction in these lyophilized powders suggests that the tertiary structures of α-lytic protease in the powder and in solution are very similar. The 15N isotropic shifts further indicate that His-57 located within the intact triad in lyophilized enzyme powders has what can be taken as a normal ‘pKa for a histidyl residue, undergoing a transition from the protonated to the neutral state with a midpoint between pH 6.0 and 7.0.

Original languageEnglish
Pages (from-to)5933-5937
Number of pages5
Issue number25
Publication statusPublished - 1984 Dec


ASJC Scopus subject areas

  • Biochemistry

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