Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

A potent radiolabeled human renin inhibitor, [3H]SR42128: enzymatic, kinetic, and binding studies to renin and other aspartic proteases

The in vitro binding of [3H]SR42128 (Iva-Phe-Nle-Sta-Ala-Sta-Arg), a potent inhibitor of human renin activity, to purified human renin and a number of other aspartic proteases was examined. SR42128 was found to be a competitive inhibitor of human renin, with a Ki of 0.35 nM at pH 5.7 and 2.0 nM at pH 7.4; it was thus more effective at pH 5.7 than at pH 7.4. Scatchard analysis of the interaction binding of [3H]SR42128 to human renin indicated that binding was reversible and saturable at both pH 5.7 and pH 7.4. There was a single class of binding sites, and the KD was 0.9 nM at pH 5.7 and 1 nM at pH 7.4. The association rate was 10 times more rapid at pH 5.7 than at pH 7.4, but there was no difference between the rates of dissociation of the enzyme-inhibitor complex at the two pHs. The effect of pH on the binding of [3H]SR42128 to human renin, cathepsin D, pepsin, and gastricsin was also examined over the pH range 3-8. All the aspartic proteases had a high affinity for the inhibitor at low pH. However, at pH 7.4, [3H]SR42128 was bound only to human renin and to none of the other aspartic proteases. Competitive binding studies with [3H]SR42128 and a number of other inhibitors on human renin or cathepsin D were used to examine the relationships between structure and activity in these systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

P-glycoprotein ATPase from the resistant pest, Helicoverpa armigera: Purification, characterization and effect of various insecticides on its transport function

Helicoverpa armigera is a major pest of agricultural crops and has developed resistance to various insecticides. A P-glycoprotein (Pgp) with ATPase activity likely to be involved in insecticide resistance was purified and characterized from insecticide-resistant H. armigera. The purification was 18-fold with 3% yield. The optimum pH and temperature were found to be 7.4 and 30-40 °C, respectively. Kinetic studies indicated that this enzyme had a K_m value of 1.2 mM for ATP. Pgp from H. armigera was partially sequenced and found to be homologous to conserved sequences of mammalian Pgps. Pesticides stimulated H. armigera Pgp ATPase activity with a maximum stimulation of up to 40%. Quenching of the intrinsic tryptophan fluorescence of purified Pgp was used to quantitate insecticide binding. Using the high-affinity fluorescent substrate, tetramethylrosamine, transport was monitored in real time in proteoliposomes containing H. armigera Pgp. The presence of Pgp could be one of the reasons for insecticide resistance in this pest.     

Thermodynamic analysis of purified human placental alpha-L-fucosidase

The temperature dependence for the hydrolysis of both 4-methylumbelliferyl-α-l-fucoside and p-nitrophenyl-α-l-fucoside was determined for purified α-l-fucosidase (EC 3.2.1.51) from human placenta. The inhibition of the enzymatic reaction by l-fucose was also studied using the first of these two substrates at different temperatures. The thermodynamic parameters calculated from the pK_m were for the 4-methylumbelliferyl-conjugate ΔF = ?6.6 kcal/mol, ΔH = ?8.5 kcal/mol, and ΔS = ?6.3 e.u. and for the p-nitrophenylconjugate ΔF = ?5.6 kcal/mol, ΔH = ?12.2 kcal/mol, and ΔS = ?21.1 e.u. The thermodynamic parameters for l-fucose were ΔH = ?12.4 kcal/mol and ΔS = ?20.1 e.u. The lower exothermicity and negative entropy calculated for the 4-methylumbelliferyl substrate compared to the thermodynamic parameters calculated for the p-nitrophenyl substrate and l-fucose suggest the existence of a secondary hydrophobic binding site for the 4-methylumbelliferyl moiety on the enzyme. The difference in the enthalpy for both substrates is also reflected in a difference in activation energy, being 15.8 kcal/mol for the 4-methylumbelliferyl substrate and 20.7 kcal/mol for the p-nitrophenyl substrate. From these results it may be concluded that altered kinetic properties of the enzyme could be the result of the binding of the “aglycone” moiety of the fluorogenic substrate to the enzyme.     

Radiochemical assay for renin utilizing a synthetic insoluble substrate

In order to provide a convenient in vitro assay for renin activity, a radiolabeled renin substrate analog, N-acetyl-Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-His-[³H]-Leu-Leu-Val-Tyr-Ser-Gly-Lys-Pro-OH, was prepared by solid-phase synthesis. The substrate peptide was bound covalently to agarose through the ?-amino group of its lysine residue. Incubation of this insoluble complex with partially purified hog renin resulted in the release of biologically active tritiated peptide into the soluble phase of the incubation mixture, at a rate proportional to the quantity of renin added. The optimum pH for cleavage was 6.5. The apparent K_m of the substrate was 1 × 10⁻⁴M, and the V_max was 83 pmoles tritiated peptide released/min/mg renin preparation added. The minimum amount of renin detectable by the assay was 2 μg, a quantity that would be expected to generate 1.0 pmole angiotensin per minute from the natural plasma substrate. Chymotrypsin, trypsin, papain, and pseudorenin, were also effective in cleaving labeled peptides from the insoluble substrate, but leucine aminopeptidase did not appear to release soluble radioactivity. The assay, as described, is useful for the measurement of large numbers of renin samples because of the speed and ease with which it may be performed. It is not yet sufficiently sensitive nor specific to measure the low levels of renin found in plasma.     

A Continuous Fluorescence Assay of Renin Activity

A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact peptide substrate. Efficient fluorescence quenching occurs as a result of favorable energetic overlap of the EDANS excited state and the DABCYL absorption, and the relatively long excited state lifetime of the EDANS fluorophore. Cleavage of the substrate by renin liberates the peptidyl-EDANS fragment from proximity with the DABCYL acceptor, restoring the higher, unattenuated fluorescence of the EDANS moiety. This leads to a time-dependent increase in fluorescence intensity, directly related to the extent of substrate consumed by renin cleavage. The kinetics of renin-catalyzed hydrolysis of this substrate have been shown to be consistent with a simple substrate inhibition model with a substrate K_m 1.5 μM at physiological pH; Cleavage of the substrate occurs specifically at the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen, as reported earlier. In this report, we describe in detail the synthesis of the fluorogenic renin substrate and its application in assays of renin activity. Assay sensitivity has been evaluated by a series of enzyme dilution experiments using the continuous assay format, showing that the assay can detect renin as low as 30 ng/ml after a incubation of only 3-5 min. It was estimated that with extended incubation time (2-3 h) the assay can detect renin at 0.5 ng/ml concentration level. An automated, high throughput fluorometric renin assay has been developed for a 96-well microtiter-plate fluorescence reader, which is useful for studies of enzyme inhibitors and enzyme stability.     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

Isomeric iodinated analogs of nimesulide: Synthesis,physicochemical characterization,cyclooxygenase-2 inhibitory activity,and transport across Caco-2 cells

Isomeric iodinated derivatives of nimesulide, with an iodine substituent on the phenoxy ring, were prepared with the aim of identifying potential candidate compounds for the development of imaging agents targeting cyclooxygenase-2 (COX-2) in the brain. Both the experimental log P_7.4 and pK_a values for these iodinated analogs were in the acceptable range for passive brain penetration. The para-iodo-substituted analog was a more potent and selective COX-2 inhibitor than nimesulide, with a potency that was comparable to the reference drug, celecoxib. Iodination at the ortho- or meta-position of the phenoxy ring was associated with a substantial loss of COX-2 inhibitory activity. Transport studies across Caco-2 cell monolayers in the presence and absence of a P-glycoprotein (P-gp) inhibitor, verapamil, indicated that the para-iodo-substituted analog was not a P-gp transport substrate; this feature is a prerequisite for potential in vivo brain imaging compounds. The para-iodo-substituted analog of nimesulide appears to be an attractive candidate for the development of radioiodine-labeled tracers for in vivo brain imaging of COX-2 levels.     

A qualitative difference in plasma renin activity in dahl rats susceptible or resistant to salt-induced hypertension

Plasma renin activity (PRA) was studied in the rats bred by Dahl for susceptibility (S-strain) or resistance (R-strain) to salt (NaCl) induced hypertension. The pH curves for PRA had different shapes. The difference in shape of the pH curves was reflected in the ratio of PRA pH 8/PRA pH 6.5. This ratio was shown to be characteristic of the strain and to be independent of changes in absolute PRA level induced by variation in dietary NaCl. The ratio of PRA pH 8/PRA pH 6.5 was also different between strains in weanling as well as adult rats. The underlying cause for the strain difference in the effect of pH on PRA is unknown, but may involve molecular differences between strains in either renin or renin substrate.     

Km values of influenza virus neuraminidases for a new fluorogenic substrate, 4-methylumbelliferone N-acetyl neuraminic acid ketoside.

A fluorogenic substrate of neuraminidase, 4-methylumbelliferone N-acetylneuraminic acid ketoside (MUN), was synthesized. K_m values were obtained for Clostridium perfringens neuraminidase and strains of A- and B-type influenza neuraminidase from chicken red blood cell influenza virus eluates.     

A kinetic characterization of the rabbit plasmin isozymes.

Steady-state and presteady-state kinetic parameters for plasmins derived from the two rabbit plasminogen isozymes have been determined. Steady-state kinetic experiments with N-α-tosyl-l-arginine methyl ester indicate that each isozyme has a similar V. Plasmin isozyme 2 has a higher K_m value for this substrate as well as a higher K_i, for the competitive inhibitor, benzamidine-HCl. Presteady-state kinetic experiments, using the p-nitrophenyl esters of p-(methylethylsulfoniummethyl)benzoate, p-(pyridiniummethyl) benzoate, p-(thiouroniummethyl)benzoate and p-(guanidinium)benzoate, indicate that each plasmin has similar rate constants of acylation (k₂). However, values of the dissociation constant (K_S) indicate that plasmin isozyme 1 has a greater binding affinity for these substrates than does isozyme 2. The magnitude of this difference varies with the substrate and is the greatest for those containing analogs of the guanidino moiety of l-arginine.     

Solvent isotope effects on α-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast α-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 °C. With p-nitrophenyl-d-glucopyranoside (pNPG), the dependence of k_cat/K_m on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, ^DOD(k_cat/K_m), of 1.9 (±0.3). The two pK_as characterizing the pH profile were increased in D₂O. The shift in pK_a2 of 0.6 units is typical of acids of comparable acidity (pK_a=6.5), but the increase in pK_a1 (=5.7) of 0.1 unit in going from H₂O to D₂O is unusually small. The initial velocities show substrate inhibition (K_is/K_m～200) with a small solvent isotope effect on the inhibition constant [^DODK_is=1.1 (±0.2)]. The solvent equilibrium isotope effects on the K_is for the competitive inhibitors d-glucose and α-methyl d-glucoside are somewhat higher [^DODK_i=1.5 (±0.1)]. Methyl glucoside is much less reactive than pNPG, with k_cat 230 times lower and k_cat/K_m 5×10⁴ times lower. The solvent isotope effect on k_cat for this substrate [=1.11 (±0. 02)] is lower than that for pNPG [=1.67 (±0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

Interaction of γ-conglutin from Lupinus albus with model phospholipid membranes: Investigations on structure,thermal stability and oligomerization status

Interaction with model phospholipid membranes of lupin seed γ-conglutin, a glycaemia-lowering protein from Lupinus albus seeds, has been studied by means of Fourier-Transform infrared spectroscopy at p²H 7.0 and at p²H 4.5. The protein maintains the same secondary structure both at p²H 7.0 and at p²H 4.5, but at p²H 7.0 a higher ¹H/²H exchange was observed, indicating a greater solvent accessibility. The difference in T_m and T_D1/2 of the protein at the abovementioned p²H's has been calculated around 20 °C. Infrared measurements have been then performed in the presence of DMPG and DOPA at p²H 4.5. DMPG showed a little destabilizing effect while DOPA exerted a great stabilizing effect, increasing the T_m of γ-conglutin at p²H 4.5 of more than 20 °C. Since γ-conglutin at p²H 4.5 is in the monomeric form, the interaction with DOPA likely promotes the oligomerization even at p²H 4.5. Interaction between DMPG or DOPA and γ-conglutin has been confirmed by turbidity experiments with DMPC:DMPG or DOPC:DOPA SUVs. Turbidity data also showed high-affinity binding of γ-conglutin to anionic SUVs made up with DOPA. The molecular features outlined in this study are relevant to address the applicative exploitation and to delineate a deeper comprehension of the natural functional role of γ-conglutin.     

Bovine factor Xa and bovine trypsin: A comparison with respect to inhibition by some amidines and guanidines

The enzymatic activity of activated bovine blood clotting factor X toward the synthetic substrate N α-benzoyl-l-arginine ethyl ester and the inhibitory effects of a series of low molecular weight synthetic aromatic amidine and guanidine compounds on that activity were studied using the steady-state kinetic method. The kinetic parameters, K_m and κ_cat, and the apparent dissociation constant K_i^′ for each inhibitor, were determined for activated factor X hydrolysis of Bz-Arg-OEt at 37 °C, pH 7.8 in 0.1 n NaCl and 0.001 m CaCl₂. The same constants were determined for bovine β-trypsin under identical conditions. Comparison of kinetic constants determined for both enzymes shows that activated factor X binds the substrate Bz-Arg-OEt less efficiently than β-trypsin by several orders of magnitude. However, binding of the inhibitors benzamidine, p-aminobenzamidine, pentamidine, M&B 4596, phenylguanidine, and p-guanidinobenzoic acid is similar for both enzymes. The results indicate that these two closely related serine proteases differ little in the structural arrangement and accessibility of the anionic “pocket” at which these inhibitors bind. The large differences observed with respect to substrate binding activity probably reflect substantial structural differences between the two enzymes at secondary sites adjacent to the primary anionic site.     

Ultrahigh and high resolution structures and mutational analysis of monomeric Streptococcus pyogenes SpeB reveal a functional role for the glycine-rich C-terminal loop

Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 Å resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC₅₀ values for trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.     

A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, K _m, k _cat, and k _cat/K _m for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters K _m, k _cat, and k _cat/K _m for Ac-nKRR-amc substrate were 100 μM, 0.112 s^?1, and 1120 M^?1·s^?1, respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.     

Purification and some properties of two polyphenol oxidases from bartlett pears

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K_m values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V_max and K_m values for oxygen and chlorogenic acid were 103 μmoles O₂ uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K_m values for chlorogenic acid were independent of pH from 3 to 7; the V_max values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K_i values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K_i values of 0.04 and 0.11 mm for enzymes A and B, respectively.     

Highly conserved glycine 86 and arginine 87 residues contribute differently to the structure and activity of the mature HIV‐1 protease

The structural and functional role of conserved residue G86 in HIV‐1 protease (PR) was investigated by NMR and crystallographic analyses of substitution mutations of glycine to alanine and serine (PR_G86A and PR_G86S). While PR_G86S had undetectable catalytic activity, PR_G86A exhibited ～6000‐fold lower catalytic activity than PR. ¹H‐¹⁵N NMR correlation spectra revealed that PR_G86A and PR_G86S are dimeric, exhibiting dimer dissociation constants (K_d) of ～0.5 and ～3.2 μM, respectively, which are significantly lower than that seen for PR with R87K mutation (K_d > 1 mM). Thus, the G86 mutants, despite being partially dimeric under the assay conditions, are defective in catalyzing substrate hydrolysis. NMR spectra revealed no changes in the chemical shifts even in the presence of excess substrate, indicating very poor binding of the substrate. Both NMR chemical shift data and crystal structures of PR_G86A and PR_G86S in the presence of active‐site inhibitors indicated high structural similarity to previously described PR/inhibitor complexes, except for specific perturbations within the active site loop and around the mutation site. The crystal structures in the presence of the inhibitor showed that the region around residue 86 was connected to the active site by a conserved network of hydrogen bonds, and the two regions moved further apart in the mutants. Overall, in contrast to the role of R87 in contributing significantly to the dimer stability of PR, G86 is likely to play an important role in maintaining the correct geometry of the active site loop in the PR dimer for substrate binding and hydrolysis. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A comparison of acylation, phosphorylation and binding in related substrates and inhibitors of serum cholinesterase

1. The K_m and catalytic-centre activities for human serum cholinesterase and methyl, ethyl, n-propyl and n-butyl butyrate substrates were determined and compared with the related inhibition constants of a similarly substituted organophosphate inhibitor series based on malaoxon. The results indicated that the catalytic-centre activities approximated to k_+2(a), the acylation rate constant, and that K_m approximated to the equilibrium binding constant. The inhibition constants measured were K_a, the equilibrium binding constant, and k_+2(p), the phosphorylation rate constant. 2. The effects of the alkyl substituents on k_+2(p) and k_+2(a) were closely parallel, and the decreasing order in each case was: n-butyl; methyl; n-propyl; ethyl. The Taft constants did not follow this order, suggesting that alkyl substituents did not primarily effect acylation or phosphorylation by electron induction. 3. For comparable homologues, the k_+2(a) values were on average 435 times the k_+2(p) values. The k_+2(p) values at 25° and pH7·6 ranged from 6·6min.⁻¹ for the diethyl member to 22·6min.⁻¹ for the di-n-butyl member. 4. The effect of the alkyl substituents on K_a and K_m were closely paralleled. The increasing order in each case was: n-butyl; n-propyl; ethyl; methyl. The K_a values were about 100 times less than the comparable K_m values. 5. Consideration of the binding energies suggested that only one of the two alkyl groups on the malaoxon homologues bound to the active site. 6. The possibility that malaoxon acted as a substrate as well as an inhibitor for cholinesterase was also investigated, but no evidence of a substrate reaction was found.     

Inhibition effect of flavonoid compounds against neuraminidase expressed in Pichia pastoris

Neuraminidase (NA) is one of the two glycoproteins on the surface of influenza virus, which cleaves terminal sialic acid residues and facilitates the release of virions from infected cells. The recombinant NA from H5N1 influenza virus strain A/Vietnam/1203/04 was expressed in Pichia pastoris X33 as a 45 kDa protein that displayed a K _m of 9.96 ± 1.26 μM with fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid. Partially purified NA was used for the inhibition and kinetic assays with eight flavonoid compounds and gallic acid. Among them, gallocatechin gallate (GCG) showed the best inhibition against NA with the IC₅₀ of 8.98 ± 0.46 μM and showed a competitive inhibition pattern with K _i value of 8.34 ± 0.25 μM. In molecular docking experiments, GCG displayed a binding energy of ?13.71 kcal/mol to the active site of NA and the galloyl moiety was required for NA inhibition activity.