Kinetically-controlled Mechanism-based Isolation Of Metabolic Serine Hydrolases In Active Form From Complex Proteomes

Serine hydrolases（SHs）constitute a large class of enzymes comprising metabolic SHs（mSHs）and serine proteases.mSHs are known for their involvement in a wide range of（patho）physiological pathways and they share a similar catalytic cycle in which a characteristic nucleophilic serine reacts with the substrate.In this work,we explored the possibility to isolate selected mSHs from complex proteomes using activity-based methods based on the kinetics between carbamate probes and mSHs.To test the principle of this approach we selected human butyrylcholinesterase（hBChE）as the model mSH and human plasma as the model proteome.The design of the carbamate probe was based on the structure of bambuterol monocarbamate（MONO）.One of the N-methyl groups of MONO was removed to increase the decarbamylation rate.The other methyl group was substituted with a flexible spacing arm which was then linked to Sepharose CL-4B.The comparison of the bimolecular carbamylation rate constant（k_I）and decarbamylation rate constant（k₃）of MONO and the carbamate probe demonstrates that the removal of one of the methyl groups increased the k₃ while the introduction of the spacing arm did not significantly modify the k_I.The performance of the method was tested by a series of single-step purification experiments.The protein isolated was analyzed by hBChE activity test,non-denaturing polyacrylamide gel electrophoresis,silver staining and BChE activity staining,Bradford assay and proteomic analysis based on UPLC-Q-TOF mass spectrometry.The results indicated that:1)hBChE binds to the probe covalently,the retention mechanism involved the active site of hBChE,and the presence of the carbamate is essential for the retention.All the active isoforms of hBChE present in human plasma（i.e.monomers,HSA-BChE adducts,dimers and tetramers）could be retained and released by the probe,differently from all the isolation methods published in the literature so far,which isolate only the tetramers.2)The incubation time,the incubation temperature,the nature and concentration of the elution agent used in the elution step were the major parameters affecting the performance.The most efficient elution buffer contained tetramethylammonium chloride（4MAC）as eluting agent.The amount of hBChE retained did not increase significantly by increasing the incubation time in the loading step,consistently with the expected high carbamylation rate,while the amount of protein contaminants showed an appreciable increase over the time.In the rinse step,warm buffer and pH 7 removed the contaminating proteins more efficiently than cold buffer and pH5.The yields and apparent purity obtained with optimized chromagraphic condition were all about 30%.An 850-fold purification factor was obtained for hBChE when a plasma aliquot was isolated using all the conditions that minimized the retention of contaminant proteins.The incubation of multiple aliquots on the same resin allowed to reach a purification factor of²500-fold,at expense of the yield.The prepared resins allowed to process up to about 8.5 mL of plasma/g of resin with constant performance.3)In addition to hBChE,the isolates also contained other three highly abundant plasma proteins tentatively identified as:serum albumin,and the immunoglobulins（Igs）IgG and IgM.According to the semi-quantitative data obtained from the proteomic analysis and the normal concentrations of hBChE,HSA and Igs in plasma,the amount of HSA and Igs retained was below 0.05%of their initial amount.Taken together,these results highlighted the high selectivity of the method and suggested that the retention of HSA and Igs could involve unstable covalent bonds,perhaps resulting from a mild reactivity of these proteins towards the probe,emphasized by their large amount in plasma.Since this method was based on the general catalytic mechanism of serine hydrolases,this work provides the basis for developing new methods for the isolation of other mSHs using carbamate probes.