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

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


Abstract 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 (3â??10). Hδ1 exchange was only dependent upon the charge state of the His57 (k ex,Im+ = 470 sâ??1, k ex,Im = 50 sâ??1), while Hε2 exchange was found to be catalyzed by hydroxide ion and phosphate base (
k\textOH - = 1.7 Ã? 1010 Mâ??1 sâ??1,
k\textHPO42 - = 1.7 Ã? 106 Mâ??1 sâ??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 Oâ??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.
  • Content Type Journal Article
  • Pages 1-16
  • DOI 10.1007/s10858-011-9527-z
  • Authors
    • Carolyn B. Lauzon, Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, USA
    • Peter van Zijl, Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, USA
    • James T. Stivers, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 314 Wood Basic Science BLD, Baltimore, MD 21205, USA

Source: Journal of Biomolecular NMR
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