Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease

Chemical Exchange Saturation Transfer (CEST) is an MRI approach that can indirectly detect exchange broadened protons that are invisible in traditional NMR spectra. We modified the CEST pulse sequence for use on high-resolution spectrometers and developed a quantitative approach for measuring exchange rates based upon CEST spectra. This new methodology was applied to the rapidly exchanging H delta 1 and H epsilon 2 protons of His57 in the catalytic triad of bovine chymotrypsinogen-A (bCT-A). CEST enabled observation of H epsilon 2 at neutral pH values, and also allowed measurement of solvent exchange rates for His57-H delta 1 and His57-H epsilon 2 across a wide pH range (3-10). H delta 1 exchange was only dependent upon the charge state of the His57 (k (ex,Im+) = 470 s(-1), k (ex,Im) = 50 s(-1)), while H epsilon 2 exchange was found to be catalyzed by hydroxide ion and phosphate base (k(OH)- = 1.7 x 10(10) M(-1) s(-1), k(HPO4)(2-) = 1.7 x 10(6) M(-1) s(-1)), reflecting its greater exposure to solute catalysts. Concomitant with the disappearance of the H epsilon 2 signal as the pH was increased above its pK (a), was the appearance of a novel signal (delta = 12 ppm), which we assigned to H gamma of the nearby Ser195 nucleophile, that is hydrogen bonded to N epsilon 2 of neutral His57. The chemical shift of H gamma is about 7 ppm downfield from a typical hydroxyl proton, suggesting a highly polarized O-H gamma bond. The significant alkoxide character of O gamma indicates that Ser195 is preactivated for nucleophilic attack before substrate binding. CEST should be generally useful for mechanistic investigations of many enzymes with labile protons involved in active site chemistry.