Fast exchanging protons are associated with many functional groups found in biological systems and are often found in the most chemically active and interesting regions of enzymes. One important example is a group of exchanging protons in the catalytic triad of the S1 family of serine proteases (MEROPS nomenclature(Rawlings and Barrett 2010
)) (). The S1 family of serine proteases includes some of the most well-known serine proteases, such as chymotrypsin, trypsin, thrombin, neuropsin, and alpha-lytic endopeptidase. These proteases are characterized by the use of a common catalytic triad consisting of an aspartic acid, a histidine and a serine (Asp102, His57, and Ser195 as found in chymotrypsinogen), that function cooperatively to help accelerate peptide bond cleavage by 1010
-fold over the non-enzymatic rate (see recent and historical reviews)(Kraut 1977
; Hedstrom 2002
; Polgar 2005
). Although the roles of S1 serine proteases are diverse, including digestion, signaling pathways(Dery et al. 1998
), immune response(Heutinck et al. 2010
), and neuronal health(Yoshida and Shiosaka 1999
), their common catalytic triad is exemplified by the widely characterized enzyme chymotrypsin, and its zymogen precursor, chymotrpysinogen.
Fig. 1 The catalytic triad of chymotrypsin/ogen and its role in peptide bond hydrolysis. (a) The catalytic triad interactions in free chymotrypsinogen at pH values below (left) and above (right) the pKa of His57-Hε2. The triad consists of a hydrogen (more ...)
Both the His57-Hε2 and Ser195-Hγ protons of chymotrypsin play central roles at multiple steps in peptide bond cleavage () (for a mechanistic review see Hedstrom (Hedstrom 2002
)), but their detection and characterization in solution has been difficult or impossible due to their rapid exchange rates with water protons. In the resting enzyme, previous NMR studies using an acyl-enzyme analogue (Robillard and Shulman 1974a
) or 15
N labeled His57 (Bachovchin 1986
) have concluded that Hγ participates in a hydrogen bond with Nε2 of neutral His57. Upon binding of the peptide substrate, Hγ is transferred to Nε2 resulting in an imidazolium ion that is stabilized by a charged hydrogen bond between His57-Hδ1 and Asp102. The shared Hδ1 proton is notable because of its extremely deshielded NMR shift (~18 ppm) (Robillard and Shulman 1974a
; Liang and Abeles 1987
). The charged state of the triad can be observed in the free state of the enzyme (or in chymotrypsinogen) by lowering the pH, which results in the same highly deshielded proton resonance attributed to Hδ1 (Robillard and Shulman 1972
; Liang and Abeles 1987
; Markley and Westler 1996
). Ser195-Hγ proton transfer also produces the essential nucleophilic alkoxide on Ser195-Oγ that attacks the carbonyl center of the peptide linkage to form the first tetrahedral intermediate. Nucleophilic attack by a catalytic serine is the mechanistic feature that characterizes all serine proteases.
During the subsequent steps of catalysis, His57-Hε2 is directly involved in three acid-base steps: (i) an acid catalysis step involving transfer of Hε2 to the amine leaving group nitrogen of the peptide, forming the acyl-enzyme intermediate, (ii) a base catalysis step involving removal of a proton from the attacking water molecule by Nε2, forming the second tetrahedral intermediate, and finally (iii) transfer of Hε2 to Ser195-Oγ to regenerate the resting enzyme. The highly dynamic nature of the Hε2 and Hγ protons, and their rapid exchange with solvent, create an important challenge for their observation in solution by NMR methods. However, their central roles in multiple steps of catalysis make such efforts highly desirable.
NMR is an important method that has contributed to our understanding of the catalytic triad. However, proton NMR studies have largely been limited to the study of the His57-Hδ1 proton because of its shielding from solvent and/or its involvement in a shared hydrogen bond with Asp102. (For a summary of NMR studies of the triad, the reader is referred to a review by Bachovchin (Bachovchin 2001
).) Chemical exchange saturation transfer (CEST) is a relatively new MRI methodology that detects exchange line broadened protons indirectly through the water resonance with enhanced sensitivity(van Zijl and Yadav 2011
; Aime et al. 2009
; Goffeney et al. 2001
; Sherry and Woods 2008
; Ward et al. 2000
). Accordingly, CEST provides a potentially useful method for the observation of rapidly exchanging amide and hydroxyl proton (Goffeney et al. 2001
; McMahon et al. 2006
; McMahon et al. 2008
) such as His57-Hε2 and Ser195-Hγ, and for the characterization of their physical properties that relate to efficient catalysis. Here we show how CEST enables NMR detection of His57-Hε2 in pH regions where previous methods have failed due to rapid solvent exchange, and also how CEST can be used to measure the pH dependence of the exchange rates for both His57-Hε2 and His57-Hδ1. Using CEST, we also observed for the first time the Hγ proton of Ser195 at neutral and basic pH values. This proton is highly deshielded in the resting enzyme at this pH range due to its hydrogen bond with His57-Nε2, indicating that the Ser195-Oγ is alkoxide-like and preactivated for nucleophilic attack in the free enzyme. CEST should prove to be a generally useful NMR method for elucidating the properties of rapidly exchanging protons in protein and nucleic acid macromolecules.