High Resolution Nuclear Magnetic Resonance Studies of the Active Site of Chymotrypsin. I. The Hydrogen Bonded Protons of the “Charge Relay” System

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin Aδ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H2O solutions but not in 2H2O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN’ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites. This proton resonance in chymotrypsin Aδ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N2-(N-acetylalanyl)-N1benzoylcarbazoyl-chymotrypsin Aδ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, -18 parts per million, to a new high pH end point of -14.4 parts per million (versus -15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen. We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin Aδ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.