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 identify the active site histidine of S. hyicus lipase we have chemically modified S. hyicus lipase with 1-bromo-octan-2-one. The enzyme is rapidly inactivated by this inhibitor with a half-time of 578 s at pH 6.5 and 30 degrees C. Addition of the enzyme's cofactor calcium increases the inactivation rate approx. 2-fold. When n-hexadecylphosphocholine, a non-hydrolysable substrate analogue, is added the inactivation rate decreases about 3-fold, suggesting that a residue in the active site of S. hyicus lipase is involved in the inactivation reaction. Inactivation of S. hyicus lipase with 14C-labelled 1-bromo-octan-2-one shows that 1.4 moles of inhibitor per mole of lipase are incorporated. The results of an electrospray mass spectrometric study of the inactivated enzyme are consistent with this finding. In order to identify the modified residue, both the inactivated and the unmodified lipase were digested with cyanogen bromide followed by trypsin. The resulting peptides were analysed using HPLC and fast atom bombardment mass spectrometry. The results allow the modified residue to be assigned to the peptide Gly597-Lys612. Collision induced dissociation mass spectrometry allowed the modified residue to be identified as His-600. From these results we conclude that this residue forms part of the catalytic triad of S. hyicus lipase.
Boots
J W
JW
Department of Enzymology and Protein Engineering, University of Utrecht, The Netherlands.
van Dongen
W D
WD
Verheij
H M
HM
de Haas
G H
GH
Haverkamp
J
J
Slotboom
A J
AJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D003488:Cyanogen Bromide; D006639:Histidine / Q000032:analysis*; D008049:Lipase / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D013210:Staphylococcus / Q000201:enzymology*; D014357:Trypsin
D006639:Histidine; D008049:Lipase; D014357:Trypsin; D003488:Cyanogen Bromide