engleski

Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase

Recently, we detected that the lipase from Streptomyces rimosus belongs to a huge but poorly characterised family of SGNH hydrolases having the alpha/beta/alpha-fold. Our biochemical characterisation is related to the specific inhibition of an extracellular lipase from Streptomyces rimosus (SRL, 24.2 kDa, Q93MW7) by preincubation method with tetrahydrolipstatin (THL). In high molar excess, THL/SRL = 590 at 25 &ordm ; ; ; C, pH = 7.0, after 2 h of incubation in an aqueous system, 56 % of the enzyme inhibition was reached, whereas at the same conditions in the presence of 50 % (v/v) 2-propanol/water 71 % enzyme inhibition was obtained. Kinetics measurements are in agreement with pseudo-first-order kinetics. The nucleophilic attack of the catalytic serine 10 of SRL results by an opening of beta-lactam 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 spectrometric experiments.

Streptomyces rimosus; extracellular SGNH-lipase; tetrahydrolipstatin; covalent inhibition; MALDI tandem mass spectrometry; ESI mass spectrometry; capillary-gel-electrophoresis-on-the-chip; kinetics

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