Contributions of NMR spectroscopy to the study of hydrogen bonds in serine protease active sites

This article is a chronology of NMR studies over the past three decades which have led to our present understanding of active site H-bonding in serine proteases. The story of H-bonding in this highly scrutinized family of enzymes, as revealed by NMR and x-ray crystallography, is to a large extent the foundation upon which our understanding of the catalytic mechanism of this archetypical enzyme system rests. Various theories have come into and out of favor as each bit of hard-earned evidence has been established, particularly with regard to the active-site imidazole, its state of protonation, its acid-base and tautomeric equilibrium constants and its H-bonding. Specific bioincorporation of N-15 and C-13 isotopic labels at the active site has proven especially useful in revealing the locations of protons and H-bond interactions through chemical shift and spin-coupling information, the latter useful also for spectral editing that reveals only the resonances of interest. However, the final chapter in the serine protease mechanism is yet to unfold. Currently competing proposals for the catalytic mechanism, one involving a low-barrier H-bond and the other a reaction driven histidine ring flip, are presented and discussed. Copyright (C) 2001 John Wiley & Sons, Ltd.