| A limiting feature of NMR is the extensive line broadening of sites undergoing fast chemical exchange with water. Chemical Exchange Saturation Transfer (CEST) is a pulse sequence that can detect rapidly exchanging protons otherwise line-broadened beyond the detection limits of traditional NMR methods. Here, we modified the CEST pulse sequence for improved use on high-resolution spectrometers, adjusted its processing methods, and developed an approach for measuring exchange rates based upon the profile of the resulting saturation difference spectrum. The new methodology was used to observe the rapidly exchanging protons in the catalytic triad of the serine protease zymogen, chymotrypsinogen-A. The catalytic triad contains three amino acids; Asp102---His57---Ser195, and two hydrogen bonds that link these three amino acids together. Three important protons to the function of the triad are Ser195-Hgamma, His57-Hepsilon2, and His57-Hdelta1. NMR spectroscopic methods have been hindered in observing His57-Hepsilon2 owing to fast exchange and line-broadening, and Ser195-Hgamma has never been detected. The His57-Hepsilon2 proton wasn't identified until 1996 and only for pH ≤ 3.5 and temperatures T ≤ 5°C. Here, a pH-titration using the new CEST methodology was collected on His57-Hdelta1 and the His57-Hepsilon2 in the catalytic triad of bovine alpha-chymotrypsinogen. The CEST-spectrum titration profile for His57-Hdelta1 matched what has been previously reported and its exchange rate was found to be constant within error across the pH range. His57-Hepsilon2 could be observed at higher pH values (pH 4.0 to 7.1) where the proton has heretofore been undetectable and its exchange rate was found to be specific base and general base shift catalyzed. Based on His57-Hepsilon2's titration profile, we propose a chemical shift assignment for Ser195-Hgamma of 12 ppm in the pH range 7.5 to 9.5. The downfield 12 ppm assignment for Hgamma reveals a Ser195-Ogamma atom that is alkoxide-like in free enzyme. The exchange rates for His57-Hepsilon2 and Ser195-Hgamma at physiological pH values, 104 s-1 and 850 s-1 respectively, are found to be catalytically competent. The results demonstrate capabilities of CEST as applied to a bio-macromolecule and provide new insights to the catalytically important and rapidly exchanging protons in the catalytic triad. |
