Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme.

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Abstract
Proton NMR spectra of serine proteases in 1H2O solutions typically show a single resonance at very low magnetic field-i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the "charge-relay system" or catalytic triad of serine proteases [Robillard, G. and Shulman, R.G. (1972) J. Mol. Biol. 71, 507-511]. Since then, there have been a number of reports that have cast doubt on its correctness. In the present work we have tested this assignment using .alpha.-lytic protease (EC 3.4.21.12, Myxobacter .alpha.-lytic proteinase), a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N.delta. of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported [Robillard, G. and Shulman, R.G. (1974) J. Mol. Biol. 86, 519-540], which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this 15N.delta.-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal.

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