Substrate Report for: BAPNA

Treatment of native psychrophilic oligopeptidase B from Serratia proteamaculans (PSP, 78 kDa) with chymotrypsin (soluble or immobilized on modified porous glass MPG-PA) in the presence of 50% glycerol leads to production of a truncated enzyme form (PSP-Chtr, ~66 kDa), which retains activity toward the low molecular weight substrate of PSP, BAPNA, but in contrast to PSP, is active toward the protein substrate azocasein. It has been shown by MALDI-TOF mass-spectrometry that PSP-Chtr lacks the N-terminal region of the molecule that envelops the catalytic domain of PSP and supposedly prevents hydrolysis of high molecular weight substrates. It has also been established that the lacking fragment corresponds to the N-terminal highest rank element of the informational structure of PSP. This finding confirms the usefulness of the method of informational structure analysis for protein engineering of enzymes. A similar treatment of PSP with immobilized trypsin also led to production of a stable truncated enzyme form (PSP-Tr, ~75 kDa) which lacked 22 C-terminal amino acid residues and completely lost enzymatic activity, presumably because of changes in the nearest environment of His652 of the catalytic triad.

Isolated from Dictamnus dasycarpus Turcz., fraxinellone exhibited multiple bioactivities against insects. In the present paper, the changes of digestive enzymes and detoxification enzymes of Mythimna separata Walker (5th instar larvae), treated with fraxinellone, were investigated. Compared with those of the control, the alpha-amylase activity of the fraxinellone-treated 5th instar larvae was inhibited, whereas the level of their protease activity was increased. Based upon further studies on the specific proteases, the levels of the active alkaline trypsin-like enzyme (BApNA as the substrate) and the chymotrypsin-like enzyme (BTEE as the substrate) activities of the treated larvae were declined; however, the level of activity of the weak alkaline trypsin-like enzyme (TAME as the substrate) of the treated ones was increased. Meanwhile, the activities of two detoxification enzymes, such as carboxylesterase (CarE) and glutathione S-transferase (GST), of the treated larvae were increased to some extent, but the activities of NADPH-P450 reductase and O-demethylase of the treated ones declined. Therefore, protease (especially the weak alkaline trypsin-like enzyme), CarE and GST played important roles in the metabolism of fraxinellone in the midgut of Mythimna separata (M. separata).

The amyloid beta (Abeta) peptide is responsible for toxic amyloid plaque formation and is central to the aetiology of Alzheimer's disease (AD). It is generated by proteolytic processing of the amyloid precursor protein (APP) by beta-secretase (BACE-1) and gamma-secretase. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach to the treatment of Alzheimer's disease. This paper reports on improved microtiter plate-based fluorescence and colorimetric assays for the high-throughput screening (HTS) of BACE-1 inhibitors achieved by employing, for the first time, casein fluorescein isothiocyanate (casein-FITC) and N-alpha-benzoyl-D,L-arginine p-nitroanilide (BAPNA) as substrates, since they are known to be readily available and convenient substrates for proteases. The methods are based on the fluorescence enhancement following casein-FITC proteolysis and the visible absorbance of the p-nitroaniline (pNA) produced by BAPNA hydrolysis, with both reactions catalysed by BACE-1. Casein-FITC is a high-affinity substrate (K (m) = 110 nM) for BACE-1, more so than the Swedish (SW) type peptide (a peptide containing the Swedish mutant of APP, a familiar mutation that enhances Abeta production). BACE-1 catalysis of casein-FITC proteolysis exhibited Michaelis-Menten kinetic. Therefore, it was found that BACE-1 was saturable with casein-FITC that was processed in a time- and pH-dependent manner with greater catalytic efficiency than observed for the SW peptide. The enantioselective hydrolysis of L-BAPNA by BACE-1 was observed. L-BAPNA was hydrolysed ten times more efficiently by BACE-1 than the WT (wild-type peptide). The novel methods were validated using a FRET assay as an independent reference method. Therefore, in order to select new leads endowed with multifunctional activities, drugs for Alzheimer's disease (AD) - potent acetylcholinesterase (AChE) inhibitors - were tested for BACE-1 inhibition using the proposed validated assays. Among these, donepezil, besides being an acetylcholinesterase inhibitor, was also found to be a BACE-1 inhibitor that displayed submicromolar potency (170 nM).

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states-closed and open-in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Treatment of native psychrophilic oligopeptidase B from Serratia proteamaculans (PSP, 78 kDa) with chymotrypsin (soluble or immobilized on modified porous glass MPG-PA) in the presence of 50% glycerol leads to production of a truncated enzyme form (PSP-Chtr, ~66 kDa), which retains activity toward the low molecular weight substrate of PSP, BAPNA, but in contrast to PSP, is active toward the protein substrate azocasein. It has been shown by MALDI-TOF mass-spectrometry that PSP-Chtr lacks the N-terminal region of the molecule that envelops the catalytic domain of PSP and supposedly prevents hydrolysis of high molecular weight substrates. It has also been established that the lacking fragment corresponds to the N-terminal highest rank element of the informational structure of PSP. This finding confirms the usefulness of the method of informational structure analysis for protein engineering of enzymes. A similar treatment of PSP with immobilized trypsin also led to production of a stable truncated enzyme form (PSP-Tr, ~75 kDa) which lacked 22 C-terminal amino acid residues and completely lost enzymatic activity, presumably because of changes in the nearest environment of His652 of the catalytic triad.

Emerald ash borer, Agrilus planipennis Fairmaire, an Asian wood-boring beetle, has devastated ash (Fraxinus spp.) trees in North American forests and landscapes since its discovery there in 2002. In this study, we collected living larvae from EAB-resistant Manchurian ash (Fraxinus mandschurica), and susceptible white (Fraxinus americana) and green (Fraxinus pennsylvanica) ash hosts, and quantified the activity and production of selected detoxification, digestive, and antioxidant enzymes. We hypothesized that differences in larval physiology could be used to infer resistance mechanisms of ash. We found no differences in cytochrome P450, glutathione-S-transferase, carboxylesterase, sulfotransferase, and tryptic BApNAase activities between larvae feeding on different hosts. Despite this, Manchurian ash-fed larvae produced a single isozyme of low electrophoretic mobility that was not produced in white or green ash-fed larvae. Additionally, larvae feeding on white and green ash produced two serine protease isozymes of high electrophoretic mobility that were not observed in Manchurian ash-fed larvae. We also found lower activity of beta-glucosidase and higher activities of monoamine oxidase, ortho-quinone reductase, catalase, superoxide dismutase, and glutathione reductase in Manchurian ash-fed larvae compared to larvae that had fed on susceptible ash. A single isozyme was detected for both catalase and superoxide dismutase in all larval groups. The activities of the quinone-protective and antioxidant enzymes are consistent with the resistance phenotype of the host species, with the highest activities measured in larvae feeding on resistant Manchurian ash. We conclude that larvae feeding on Manchurian ash could be under quinone and oxidative stress, suggesting these may be potential mechanisms of resistance of Manchurian ash to EAB larvae, and that quinone-protective and antioxidant enzymes are important counter-adaptations of larvae for dealing with these resistance mechanisms.

Isolated from Dictamnus dasycarpus Turcz., fraxinellone exhibited multiple bioactivities against insects. In the present paper, the changes of digestive enzymes and detoxification enzymes of Mythimna separata Walker (5th instar larvae), treated with fraxinellone, were investigated. Compared with those of the control, the alpha-amylase activity of the fraxinellone-treated 5th instar larvae was inhibited, whereas the level of their protease activity was increased. Based upon further studies on the specific proteases, the levels of the active alkaline trypsin-like enzyme (BApNA as the substrate) and the chymotrypsin-like enzyme (BTEE as the substrate) activities of the treated larvae were declined; however, the level of activity of the weak alkaline trypsin-like enzyme (TAME as the substrate) of the treated ones was increased. Meanwhile, the activities of two detoxification enzymes, such as carboxylesterase (CarE) and glutathione S-transferase (GST), of the treated larvae were increased to some extent, but the activities of NADPH-P450 reductase and O-demethylase of the treated ones declined. Therefore, protease (especially the weak alkaline trypsin-like enzyme), CarE and GST played important roles in the metabolism of fraxinellone in the midgut of Mythimna separata (M. separata).

The amyloid beta (Abeta) peptide is responsible for toxic amyloid plaque formation and is central to the aetiology of Alzheimer's disease (AD). It is generated by proteolytic processing of the amyloid precursor protein (APP) by beta-secretase (BACE-1) and gamma-secretase. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach to the treatment of Alzheimer's disease. This paper reports on improved microtiter plate-based fluorescence and colorimetric assays for the high-throughput screening (HTS) of BACE-1 inhibitors achieved by employing, for the first time, casein fluorescein isothiocyanate (casein-FITC) and N-alpha-benzoyl-D,L-arginine p-nitroanilide (BAPNA) as substrates, since they are known to be readily available and convenient substrates for proteases. The methods are based on the fluorescence enhancement following casein-FITC proteolysis and the visible absorbance of the p-nitroaniline (pNA) produced by BAPNA hydrolysis, with both reactions catalysed by BACE-1. Casein-FITC is a high-affinity substrate (K (m) = 110 nM) for BACE-1, more so than the Swedish (SW) type peptide (a peptide containing the Swedish mutant of APP, a familiar mutation that enhances Abeta production). BACE-1 catalysis of casein-FITC proteolysis exhibited Michaelis-Menten kinetic. Therefore, it was found that BACE-1 was saturable with casein-FITC that was processed in a time- and pH-dependent manner with greater catalytic efficiency than observed for the SW peptide. The enantioselective hydrolysis of L-BAPNA by BACE-1 was observed. L-BAPNA was hydrolysed ten times more efficiently by BACE-1 than the WT (wild-type peptide). The novel methods were validated using a FRET assay as an independent reference method. Therefore, in order to select new leads endowed with multifunctional activities, drugs for Alzheimer's disease (AD) - potent acetylcholinesterase (AChE) inhibitors - were tested for BACE-1 inhibition using the proposed validated assays. Among these, donepezil, besides being an acetylcholinesterase inhibitor, was also found to be a BACE-1 inhibitor that displayed submicromolar potency (170 nM).