IDENTIFICATION OF THE ACTIVE-SITE HISTIDINE IN STAPHYLOCOCCUS-HYICUS LIPASE USING CHEMICAL MODIFICATION AND MASS-SPECTROMETRY

Staphylococcus hyicus lipase is a serine hydrolase. In order to identi fy the active site histidine of S. hyicus lipase we have chemically mo dified S. hyicus lipase with 1-bromo-octan-2-one. The enzyme is rapidl y inactivated by this inhibitor with a half-time of 578 s at pH 6.5 an d 30 degrees C. Addition of the enzyme's cofactor calcium increases th e inactivation rate approx. 2-fold. When n-hexadecylphosphocholine, a non-hydrolysable substrate analogue, is added the inactivation rate de creases about 3-fold, suggesting that a residue in the active site of S. hyicus lipase is involved in the inactivation reaction. Inactivatio n of S. hyicus lipase with C-14-labelled 1-bromo-octan-2-one shows tha t 1.4 moles of inhibitor per mole of lipase are incorporated. The resu lts of an electrospray mass spectrometric study of the inactivated enz yme are consistent with this finding. In order to identify the modifie d residue, both the inactivated and the unmodified lipase were digeste d with cyanogen bromide followed by trypsin. The resulting peptides we re analysed using HPLC and fast atom bombardment mass spectrometry. Th e results allow the modified residue to be assigned to the peptide Gly (597)-Lys(612). Collision induced dissociation mass spectrometry allow ed the modified residue to be identified as His-600. From these result s we conclude that this residue forms part of the catalytic triad of S . hyicus lipase.