Phosphorus-31 NMR and mass spectrometry of atropinesterase and some serine proteases phosphorylated with a transition-state analog

Abstract

The serine residue in the active center of atropinesterase (AtrE), alpha-chymotrypsin (Chymo), and subtilisin A (Sub) and in alpha-chymotrypsinogen (Chymogen) was labeled with a diisopropylphosphoryl (DP) group. The labeled proteins were studied in buffered aqueous solution under various native and denaturing conditions with 31P NMR before and after being subjected to "ageing", a process leading to conversion of the DP group into a monoisopropylphosphoryl (MP) group. Besides, the model compounds Gly-Ser(DP), Gly-Glu-Ser(DP)-Gly-OEt, and diisopropyl hydrogen phosphate were investigated under similar conditions and in other solvents with different hydrogen-bonding capacity. Mass spectrometry was used to analyze products resulting from ageing in the presence of H2(18)O. The 31P chemical shift of the DP proteins increases according to a simple titration curve upon lowering the pH from 9.0 to 5.0. This is ascribed to protonation of a particular histidine residue in the active center that interacts with a nearby isopropoxy group by hydrogen bonding with the ester oxygen. In DP-AtrE, hydrogen bonding at the phosphoryl oxygen dominates the interaction between substituent and protein; in the other DP proteins, nonbonding interactions become more dominant in the order Chymogen less than Chymo less than Sub. DP-AtrE, DP-Chymo, and DP-Sub age according to first-order kinetics. The pH dependence of the reaction rate constant ka indicates that ageing is catalyzed by the protonated histidine, which is responsible for the increase in chemical shift. The direct interaction between the phosphoryl group and the histidine is lost upon ageing whereas there is an increase in the nonbonding interaction of the remaining isopropyl group with the protein in the order Chymo less than Sub less than AtrE. The maximum value of ka when the histidine is fully protonated (kam) increases in the same order. Ageing of the DP enzymes occurs exclusively by C-O fission, yielding 2-propanol and propene. Since the amount of 2-propanol decreased and that of propene increased in the order Chymo to Sub to AtrE, the increase in kam has been interpreted as a shift in character of ageing from mainly SN2 for Chymo to considerably SN1 for AtrE and Sub. This has been attributed to preferential stabilization of the SN1 transition state by an interplay of hydrogen-bonding and nonbonding interactions between the phosphoryl group and the protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal Structures of Brain Group-VIII Phospholipase A2 in Nonaged Complexes with the Organophosphorus Nerve Agents Soman and Sarin,

“…Aging rates vary over a wide range; however, aged complexes had always been observed for X-ray structural determinations prior to the recent work on human CE1 (9) and the present work. While no quantitative study was conducted, the results presented here suggest that the half-life for aging in the gVIII-PLA2:sarin complex, which was solved using crystals that were incubated at room temperature for 2 weeks, is significantly longer than the 10 day half-life seen for the chymotrypsin-DFP complex (13). …”

“…Aging rates have a complex dependence on the specific enzyme-OP inhibitor system, pH, and temperature. Measured aging half-lives of enzyme-OP inhibitor complexes range from 10 days for the chymotrypsin-DFP complex (13) to 1 minute for the AChE-soman complex (11). Typically, aging is a slow process that is enhanced in the case of the human AChE and BChE, which share 55% sequence identity.…”

Engineering resistance to ‘aging’ of phosphylated human acetylcholinesterase Role of hydrogen bond network in the active center

“…In general, the catalysis of aging is particularly efficient in ChE's compared to OPconjugates with other serine hydrolases [12,13], indicating a specific involvement of residues vicinal to the phosphyl moiety in the ChE active center.…”

Use of NMR techniques for toxic organophosphorus compound profiling

“…31 P NMR spectroscopy has been indispensable in studies of the serine hydrolase inhibition induced by OP compounds. The range of application covers structural characterization of OP-chymotrypsin conjugates [31][32][33][34][35], pH titration studies of (diisopropylphosphoryl)serine proteinases [36], aging studies of atropinesterase and several serine proteases [37], and indirect calcium-binding site characterization of trypsin, chymotrypsin, and subtilisin [38]. Segall et al [39] characterized the OP compound moiety of phosphorylated cholinesterases by both direct and comparative 31 P NMR spectroscopy.…”