News & Updates
Search Research Content
Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease.
|Title||Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease.|
|Publication Type||Journal Article|
|Year of Publication||2011|
|Authors||Lauzon CB, van Zijl P, Stivers JT|
|Journal||Journal of biomolecular NMR|
|Date Published||2011 Aug|
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 Hδ1 and Hε2 protons of His57 in the catalytic triad of bovine chymotrypsinogen-A (bCT-A). CEST enabled observation of Hε2 at neutral pH values, and also allowed measurement of solvent exchange rates for His57-Hδ1 and His57-Hε2 across a wide pH range (3-10). Hδ1 exchange was only dependent upon the charge state of the His57 (k (ex,Im+) = 470 s(-1), k (ex,Im) = 50 s(-1)), while Hε2 exchange was found to be catalyzed by hydroxide ion and phosphate base (k(OH)⁻ = 1.7 × 10(10) M(-1) s(-1), K(HPO)²⁻₄ = 1.7 × 10(6) M(-1) s(-1)), reflecting its greater exposure to solute catalysts. Concomitant with the disappearance of the Hε2 signal as the pH was increased above its pK (a), was the appearance of a novel signal (δ = 12 ppm), which we assigned to Hγ of the nearby Ser195 nucleophile, that is hydrogen bonded to Nε2 of neutral His57. The chemical shift of Hγ is about 7 ppm downfield from a typical hydroxyl proton, suggesting a highly polarized O-Hγ bond. The significant alkoxide character of Oγ 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.
|Alternate Journal||J. Biomol. NMR|