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Protein Science (2003), 12:794-810.
Copyright © 2003 The Protein Society

Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN' by NMR

Regina M. Day1, Craig J. Thalhauser2, James L. Sudmeier2, Matthew P. Vincent2, Ekaterina V. Torchilin2, David G. Sanford2, Christopher W. Bachovchin2 and William W. Bachovchin2

1 Pulmonary and Critical Care Division, Department of Medicine, New England Medical Center/Tupper Research Institute, Boston, Massachusetts 02111, USA
2 Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA

Reprint requests to: William W. Bachovchin, Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111, USA; e-mail: william.bachovchin{at}tufts.edu; fax (617) 636-2409.

We have determined by 15N, 1H, and 13C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every 15N, 1H, C{varepsilon}1, and C{delta}2 resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pKa = 7.30 ± 0.03 at 25°C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pKa value of 7.9 ± 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C{varepsilon}1-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C{varepsilon}1-H. . .O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N{delta}1-H tautomer, exhibiting 13C{delta}1 chemical shifts ~9 ppm higher than those for N{varepsilon}2-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by 15N-1H NOE effects, and titrates with rapid proton exchange kinetics linked to a pKa value of 7.47 ± 0.02.

Keywords: Histidine; tautomerism; serine protease; hydrogen bond; NMR spectroscopy; peptide boronic acid inhibitor; 15N NMR


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