Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase.
We have recently detected that the lipase from Streptomyces rimosus belongs to a large but poorly characterised family of SGNH hydrolases having the alpha beta alpha-fold. Our biochemical characterisation relates to the specific inhibition of an extracellular lipase from Streptomyces rimosus (SRL, 24.2 kDa, Q93MW7) by the preincubation method with tetrahydrolipstatin (THL). In high molar excess (THL/SRL=590 at 25 degrees C, pH=7.0) and after 2 h of incubation in an aqueous system, 56% of the enzyme inhibition was reached. Under the same conditions and in the presence of 50% (v/v) 2-propanol/water, 71% enzyme inhibition was obtained. Kinetic measurements are in agreement with pseudo-first-order kinetics. The nucleophilic attack of the catalytic serine residue 10 of SRL occurs via an opening of the beta-lactone ring of tetrahydrolipstatin and formation of a covalent ester bond. The intact covalent complex of SRL-inhibitor was analysed by ESI and vacuum MALDI mass spectrometry and, furthermore, the exact covalent THL linkage was determined by vacuum MALDI high-energy collision-induced dissociation tandem mass spectrometry.
Asler
Ivana Lescić
IL
Rudjer Bosković Institute, POB 180, 10002 Zagreb, Croatia.
Zehl
Martin
M
Kovacić
Filip
F
Müller
Roland
R
Abramić
Marija
M
Allmaier
Günter
G
Kojić-Prodić
Biserka
B
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D019075:Electrophoresis, Capillary; D007783:Lactones / Q000494:pharmacology; D008025:Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000077403:Orlistat; D012694:Serine; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000201:enzymology*; D014357:Trypsin
D001426:Bacterial Proteins; D007783:Lactones; D012694:Serine; D000077403:Orlistat; D014357:Trypsin; D008025:Ligases