STRUCTURE OF GAMMA-CHYMOTRYPSIN IN THE RANGE PH 2.0 TO PH 10.5 SUGGESTS THAT GAMMA-CHYMOTRYPSIN IS A COVALENT ACYL ENZYME ADDUCT AT LOW PH

Crystals of gamma-chymotrypsin (gamma-CHT) grown at pH 7.0 are stable from pH 2.0 to 11.0. Crystalline gamma-CHT therefore provides an unusually favourable system to observe the structure of a protein and its bound solvent over a broad range of pH. In this report we describe the high-resolution refined structure of gamma-CHT at pH values of 2.0, 7.0 and 10.5. The apparent tetrapeptide seen bound in the active site of gamma-CHT at pH 7.0 is also present at pH 2.0 and 10.5 although it is better defined at low pH. A comparison of the respective structures shows that there is additional electron density in the low pH structure at the point where the side-chain of Ser 195 approaches most closely to the presumptive inhibitor. This suggests that the adduct is most likely to be covalently linked to the enzyme at low pH and to be non-covalent at higher pH. As the pH is lowered from 7.0 to 2.0, the side-chain of His 40 rotates approximately 120-degrees about its C-alpha-C-beta bond and, in concert, the side-chain of Gln 34 also rotates approximately 140-degrees about its C-alpha-C-beta bond. Apart from these localized rearrangements in the vicinity of His 40, the structure of gamma-CHT at pH 2.0 is very similar to that at neutral pH. The structure of gamma-CHT at pH 10.5 is also seen to be almost identical with that at neutral pH. There is no indication that the internal salt bridge between Asp 194 and the alpha-amino group of lle 16 begins to dissociate at pH 10.5. With the exception of the vicinity of His 40, the structure of the bound solvent in the crystal structures at low, neutral and high pH is very similar. There is no clearcut example where the solvent is seen to rearrange in response to the titration of a group on the surface of the protein.