caa a22 4500 445325844 CHVBK 20180317142720.0 cr unu---uuuuu 170323e20110801xx s 000 0 eng 10.1007/s10858-011-9527-z doi (NATIONALLICENCE)springer-10.1007/s10858-011-9527-z Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease [Elektronische Daten] [Carolyn Lauzon, Peter van Zijl, James Stivers] 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+=470s−1, k ex,Im=50s−1), while Hε2 exchange was found to be catalyzed by hydroxide ion and phosphate base ( $$ k_{{{\text{OH}}^{ - } }} $$ =1.7×1010M−1s−1, $$ k_{{{\text{HPO}}_{4}^{2 - } }} $$ =1.7×106M−1s−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=12ppm), 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 7ppm 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. Springer Science+Business Media B.V., 2011 Catalytic triad nationallicence CEST nationallicence Chemical exchange nationallicence Saturation transfer nationallicence Chymotrypsinogen nationallicence Fast exchange nationallicence Serine protease nationallicence Lauzon Carolyn Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, 21205, Baltimore, MD, USA aut van Zijl Peter Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, 21205, Baltimore, MD, USA aut Stivers James Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 314 Wood Basic Science BLD, 21205, Baltimore, MD, USA aut Journal of Biomolecular NMR Springer Netherlands 50/4(2011-08-01), 299-314 0925-2738 50:4<299 2011 50 10858 https://doi.org/10.1007/s10858-011-9527-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9527-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lauzon Carolyn Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, 21205, Baltimore, MD, USA aut NATIONALLICENCE 700 1- van Zijl Peter Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, 21205, Baltimore, MD, USA aut NATIONALLICENCE 700 1- Stivers James Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 314 Wood Basic Science BLD, 21205, Baltimore, MD, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 50/4(2011-08-01), 299-314 0925-2738 50:4<299 2011 50 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer