Nitrogen-15 nuclear magnetic resonance spectroscopy. The state of histidine in the catalytic triad of .alpha.-lytic protease. Implications for the charge-relay mechanism of peptide-bond cleavage by serine proteases

Abstract

Histidine, enriched in ^(15)N in the imidazole ring, has been incorporated into the "catalytic triad" of serinyl. histidyl, and aspartyl residues of α-lytic protease, using a histidine auxotroph of myxobacter 495. The pK_a of this histidyl residue is 7.0 ± 0.1 at 26 ºC, as determined by the changes of its ^(15)N chemical shifts in nuclear magnetic resonance spectra. This finding is contrary to previously published reports that histidyl residues at the active sites of serine proteases are likely to be abnormally weak bases, while the "buried" aspartyl residues of the catalytic triads are likely to be abnormally weak acids and thus offers no support for the current formulation of the charge-relay mechanism of action of serine proteases. The ^(15)N chemical shifts further demonstrate that, at catalytically active pH values, the tautomer with hydrogen on N3 (the π nitrogen) of the imidazole ring predominates. This is an unusual tautomeric state both for simple 4-substituted imidazole derivatives and for histidyl residues in proteins, and its predominance in ɑ-lytic protease can be reasonably attributed to a hydrogen-bonded interaction between NH at the 3 position and the adjacent "buried" carboxylate group of aspartic acid. The implication of the results for the respective roles of the aspartyl and histidyl residues in serine protease catalysis is discussed in terms of the energy requirements of the individual steps for the formation and breakdown of the serine ester intermediate.

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