Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme.
Bachovchin, W W
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. & 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 184.108.40.206, Myxobacter alpha-lytic proteinase), a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N delta 1 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. & 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 1-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal.
ACESSO AO ARTIGOhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=390887
- Droplet Size Distribution in Water-Crude Oil Emulsions by Low-Field NMR
- Evaluation of the anisotropy field for fine-particle systems from low-field thermomagnetic curves
- Zero- to low-field MRI with averaging of concomitant gradient fields
- Nitrogen-15 nuclear magnetic resonance of aliphatic tripeptides.
- NITROGEN-15 MAGNETIC RESONANCE SPECTROSCOPY, I. CHEMICAL SHIFTS*