KMI-008, a novel beta-secretase inhibitor containing a hydroxymethylcarbonyl isostere as a transition-state mimic: design and synthesis of substrate-based octapeptides

A novel class of substrate-based β-secretase (BACE1) inhibitors containing a hydroxymethylcarbonyl (HMC) isostere was designed and synthesized. Phenylnorstatine [(2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid; Pns] was an effective transition-state mimic at the P₁ position. Structure–activity relationships (SARs) of the P₃–P₃′ positions of BACE1 inhibitors were studied.     

Design and synthesis of statine-containing BACE inhibitors

Utilizing structure-based techniques and solid-phase synthesis, statine-based tetrapeptide BACE inhibitors were designed and synthesized using a heptapeptide BACE transition-state mimetic, 1, as the starting point. Structure–activity relationship studies at the P₃, P₂, and P₂′ positions as well as the N-terminal capping group on scaffold 5 led to the discovery of potent inhibitors 27, 32, and 34 (IC₅₀ <100 nM). In addition, computational analysis and the X-ray structure of BACE–inhibitor 38 are discussed.     

Nonionic detergent-induced activation of an esterase from Bacillus megaterium 20-1

An esterase-producing Bacillus megaterium strain (20-1) was isolated from a soil sample collected in South Korea. The cloned gene showed that the esterase 20-1 composed of 310 amino acids corresponding to a molecular mass (M_r) of 34,638. Based on the M_r and the protein sequence, the esterase 20-1 belonged to the H lipase/esterase group. The optimum temperature and pH of the purified His-tagged enzyme were 20–35 °C and 8.0, respectively. The esterase 20-1 showed a ‘nonionic detergent-induced activation’ phenomenon, which was a detergent type- and concentration-dependent process. In comparison with the native enzyme, the Tween 80-treated enzyme had relatively a similar k_cat value of 274 s⁻¹ but a very low K_m value of 0.037 mM for PNPC (C₆), therefore, it showed a 14-fold increase in k_cat/K_m value.     

Hydrolysis of fibrinogen and plasminogen by immobilized earthworm fibrinolytic enzyme II from Eisenia fetida

Earthworm fibrinolytic enzyme II (EFE-II) from Eisenia fetida has a broad hydrolytic specificity for peptide bonds. Our experiments show that EFE-II can hydrolyze the specific chromogenic substrates of thrombin (Chromozym TH), trypsin (Chromozym TRY) and elastase (Chromozym ELA). The Michaelis–Menten constant (K_m) for Chromozym ELA (245 μM) is much higher than those for the thrombin (90 μM) and trypsin (60 μM) substrates. On the other hand, EFE-II is inhibited most strongly by soybean trypsin inhibitor (SBTI), and weakly inhibited by elastinal, suggesting that EFE-II has a trypsin-like activity. Degradation of plasminogen (PLg) and fibrinogen by EFE-II was investigated after EFE-II had been immobilized onto 1,1′-carboryl-diimidazole (CDI)-activated Sepharose CL-6B. The immobilized EFE-II has 55–60% activity of the native enzyme with a higher thermal and pH resistance. EFE-II cleaves PLg at four hydrolytic sites: Lys₇₇–Arg₇₈, Arg₃₄₂–Met₃₄₃, Ala₄₄₄–Ala₄₄₅ and Arg₅₅₇–Ile₅₅₈. The site Arg₅₅₇–Ile₅₅₈ is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala₄₄₄–Ala₄₄₅ released mini-plasmin with secondary activity to hydrolyze fibrin. Immobilized EFE-II degrades not only the A chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in the N-terminal region at the Val₂₁–Glu₂₂ site.     

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae

Polyphosphates of different chain lengths (P₃, P₄, P₁₅, P₃₅), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p₄A and P₄ (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap₄A was not inhibitory. P₄ and P₁₅ were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P₄ and P₁₅ behaved as competitive inhibitors, with K_i values of 0.5 μM and 0.2 μM, respectively. In addition, P₄ (at 1 μM) and P₁₅ (at 0.3 μM) changed the Hill coefficient (n_H) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P₄ and P₁₅ decreased V and modified only slightly the K_m values of the enzyme towards tRNA.     

Site-directed mutagenesis of yeast phosphoglycerate kinase Arginines 65, 121 and 168

In the absence of a structure of the closed form of phosphoglycerate kinase we have modified by site directed mutagenesis several of the residues which, on the basis of the open form structure, are likely to be involved in substrate binding and catalysis. Here we report on the kinetic and anion activation properties of the yeast enzyme modified at positions 65, 121 and 168. In each case an arginine, thought to be involved in the binding of the sugar substrate's non-transferable phosphate group, has been replaced by lysine (same charge) and by methionine (no charge). K_m values for 3-phosphoglycerate of all six mutant enzymes are only marginally higher than that of the wild-type enzyme. Removing the charge associated with two of the three arginine residues appears to influence (as judged by the measured K_m's) the binding of ATP. Although binding affinity is not necessarily coupled to turnover the substitutions which have the greatest effect on the K_m's do correlate with the reduction in enzymes maximum velocity. The one exception to this generalisation is the R65K mutant which, surprisingly, has a significantly higher k_cat than the wild-type enzyme. In the open form structure of the pig muscle enzyme each of the three substituted arginines residues are seen to make two hydrogen bonds to the sugar substrate's non-transferable phosphate. From this it might be expected that anion activation would be similarly affected by the substitution of any one of these three residues. Although the interpretation of such effects are complicated by the fact that one of the mutants (R65M) unfolds at low salt concentrations, this appears not to be the case. Replacing Arg¹²¹ and Arg¹²¹ with methionine reduces the anion activation whereas a lysine in either of these two positions practically destroys the effect. With the substitutions at residue 65 the opposite is observed in that the lysine mutant shows anion activation whereas the methionine mutant does not.     

Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest

Two thermophilic extracellular proteases, designated Lmm-protease-Lh (29 kDa) and Hmm-protease-Lh (62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK₂. Both novel proteases maintained full activity at 60 °C after 1-h incubation at 10 °C as well as at 80 °C, showing optimum k_cat/K_m values at pH 7.00 and 60 °C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis–Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis–Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k_cat/K_m) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis–Menten parameters.     

Use of a 49-peptide library for a qualitative and quantitative determination of pseudomonal serralysin specificity

The Pseudomonas aeruginosa serralysin (E.C. 3.4.24.40.), which is a zinc-dependent metalloprotease from the metzincin superfamily, has quite a broad specificity, which has not yet been clearly identified. We have studied it with an original approach, using a 49-peptide library of the type Z–AXXA (amide) (X=A, L, V, F, S, R, E). The library was analyzed by LC-MS before and after enzymatic hydrolysis. A great number of hydrolyzed peptides were screened and the preferential hydrolysis was the X–X peptide bond, even if in some cases, A–X and X–A bond could be hydrolyzed. No amino acids with a ionized side chain could be found in the P₁′ position. The results obtained suggest that the specificity in the P_n′ position, where an hydrophobic residue was preferentially found, seems more selective that in the P_n position. The P₁ position was not very specific, but, on a quantitative point of view, the enzymatic activity was particularly increased when R, F or A were in this position. The results allow us to define the P₁′ and P₁ residues for an optimal substrate of pseudomonal serralysin and usable for the design and the synthesis of a specific inhibitor.     

The Mechanism of Salt Activation of Thermolysin: Relation with Pressure Activation and Implications of Hydration Change Coupled with Rate Process

Thermolysin (E.C. 3.4.24.4) shows a remarkable increase in catalytic activity at elevated salt concentrations or hydrostatic pressures. Salt effected K_cat, only, whilst the effect of pressure was related to both K_cat, and K_m. The turnover, derived from k_cat/K_m(V), of the hydrolysis of an N-acyldipeptide amide substrate was scarcely affected by addition of salt. These results were interpreted in terms of the stabilization of increased (or exposed) charges at the transition state of the reaction.     

A substitutive substrate for measurements of beta-ketoacyl reductases in two fatty acid synthase systems

Bacterial β-ketoacyl-ACP reductase (FabG) and the β-ketoacyl reductase domain in mammalian fatty acid synthase (FAS) have the same function and both are rendered as the novel targets for drugs. Herein we developed a convenient method, using an available compound ethyl acetoacetate (EAA) as the substitutive substrate, to measure their activities by monitoring decrease of NADPH absorbance at 340 nm. In addition to the result, ethyl 3-hydroxybutyrate (EHB) was detected by HPLC analysis in the reaction system, indicating that EAA worked effectively as the substrate of FabG and FAS since its β-keto group was reduced. Then, the detailed kinetic characteristics, such as optimal ionic strength, pH value and temperature, and kinetic parameters, for FabG and FAS with this substitutive substrate were determined. The K_m and k_cat values of FabG obtained for EAA were 127 mM and 0.30 s^− 1, while those of this enzyme for NADPH were 10.0 μM and 0.59 s^− 1, respectively. The corresponding K_m and k_cat values of FAS were 126 mM and 4.63 s^− 1 for EAA; 8.7 μM and 4.09 s^− 1 for NADPH. Additionally, the inhibitory kinetics of FabG and FAS, by a known inhibitor EGCG, was also studied.     

Uptake and light activated esterification of P by isolated chloroplasts

1. 1. Esterification of ³²P₁ by illuminated chloroplasts prepared on a sucrose gradient was examined to establish the optimal incubation conditions.

2. 2. The evidence is consistent with phosphorylation being closely coupled to the sum of noncyclic and pseudocyclic electron flow and with the rate of electron flow responding to the availability of electron acceptors.

3. 3. Apparent K_m values for ADP and Mg²⁺ were found to be 40 and 250 μM, respectively. The K_m value for Mg²⁺ was increased by the presence of Ca²⁺. Two apparent values were observed for P₁ at 0.2 and 1.1 mM. Chloroplast damage resulted in increased apparent K_m (P₁) values.

4. 4. Acceleration of the esterification resulting from the addition of ADP and P₁ to the medium indicated that these compounds were able to penetrate to the active site of esterification.

5. 5. Ribose 5-phosphate (Rib-5-P) was shown to inhibit P₁ esterification without affecting the apparent K_m for ADP or P₁. The evidence suggests that Rib-5-P interferes with the uptake of P₁, and possibly ADP.

Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase of the tobacco hornworm (Manduca sexta: Sphingidae)

A new class of inhibitors of juvenile hormone epoxide hydrolase (JHEH) of Manduca sexta and further in vitro characterization of the enzyme are reported. The compounds are based on urea and amide pharmacophores that were previously demonstrated as effective inhibitors of mammalian soluble and microsomal epoxide hydrolases. The best inhibitors against JHEH activity so far within this class are N-[(Z)-9-octadecenyl]-N′-propylurea and N-hexadecyl-N′-propylurea, which inhibited hydrolysis of a surrogate substrate (t-DPPO) with an IC₅₀ around 90 nM. The importance of substitution number and type was investigated and results indicated that N, N′-disubstitution with asymmetric alkyl groups was favored. Potencies of pharmacophores decreased as follows: amide>urea>carbamate>carbodiimide>thiourea and thiocarbamate for N, N′-disubstituted compounds with symmetric substituents, and urea>amide>carbamate for compounds with asymmetric N, N′-substituents. JHEH hydrolyzes t-DPPO with a K_m of 65.6 μM and a V_max of 59 nmol min⁻¹ mg⁻¹ and has a substantially lower K_m of 3.6 μM and higher V_max of 322 nmol min⁻¹ mg⁻¹ for JH III. Although none of these compounds were potent inhibitors of hydrolysis of JH III by JHEH, they are the first leads toward inhibitors of JHEH that are not potentially subject to metabolism through epoxide degradation.     

Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete

A thermostable laccase was isolated from an unidentified fungal isolate [Enz. Microb. Technol. 33 (2003) 212], and tentatively named UD4. This work indicates that the enzyme has unique properties other than its thermostability. Investigation into the kinetic parameters of the thermostable laccase yielded an unusually high affinity for ABTS as a substrate (low K_m) when compared with available published data for other laccase isozymes. The specificity constant (k_cat/K_m) was found to be considerably higher than laccase from other sources and is comparable to “white” laccase from Pleurotus ostreatus (POXA1). However, POXA1 isozyme exhibits a large turnover number (k_cat) that contributes to its high specificity constant whereas the high specificity constant for UD4 laccase is achieved by having a high substrate affinity. The UD4 thermostable laccase, like most other laccases, is able to utilize guaiacol as a substrate, whereas POXA1 is unable to oxidize guaiacol, indicating a broader substrate range for the thermostable laccase from UD4. The thermostable laccase is inhibited by sodium azide through non-competitive inhibition, and by thioglycolic acid and hydroxylamine through competitive inhibition. The high specificity constant, substrate affinity and broader substrate range of the thermostable laccase from UD4 indicates that it is a highly favourable candidate enzyme for industrial application.     

Use of 3-D computer modelling and kinetic studies to analyse grapefruit pyrophosphate-dependent phosphofructokinase

The glycolytic reaction of grapefruit PPi-dependent phosphofructokinase (PFP) depends on the presence of Fru-2,6-P₂ (K_a=6.7 nM). This molecule was further demonstrated in grapefruit juice sac cells. Citrate, -ketoglutarate and isocitrate competitively inhibited the binding of Fru-2,6-P₂ to PFP. The affinity for Fru-6-P (K_m=159 μM) and PPi (K_m=33 μM) were not affected by the addition of these molecules. In the gluconeogenic reaction, the presence of Fru-2,6-P₂ did not affect the K_m of Fru-1,6-P₂ (61 μM) in contrast to orange fruit PFP. These results led to the building of a computer model of PFP, based on the known structure of Bacillus stearothermophilus ATP-dependent phosphofructokinase (ATP-PFK). The results show that catalysis of Fru-6-P in the chain is most unlikely, due to amino-acid substitutions and that Fru-2,6-P₂ can bind between the and β subunits.     

Isolation of a renin-like enzyme from the leech Theromyzon tessulatum

This article reports the purification of a renin-like enzyme (an aspartyl protease) from head parts of the leech Theromyzon tessulatum. After four steps of purification including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC, this enzyme was purified to homogeneity. The renin-like enzyme (of 32 kDa) hydrolyses at neutral pH and at 37°C, the Leu¹⁰-Leu¹¹ bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu¹¹-Val¹²-Tyr¹³-Ser¹⁴ peptide as products, with a specific activity of 1.35 pmol AI/min/mg (K_m 22 μM; K_cat 2.7). The hydrolysis of angiotensinogen is inhibitable at 90% by pepstatin A (IC₅₀ = 4.6 μM), consistent with a renin activity. This is the first biochemical evidence of renin-like enzyme in invertebrates.     

1-Methoxy-, 1-deoxy-11-hydroxy- and 11-hydroxy-1-methoxy-Delta(8)-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Three series of new cannabinoids were prepared and their affinities for the CB₁ and CB₂ cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ⁸-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB₂ receptor than for the CB₁ receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ⁸-THC (JWH-229, 6e) has effectively no affinity for the CB₁ receptor (K_i=3134±110 nM) and high affinity for CB₂ (K_i=18±2 nM).     

Enhanced thermostability and tolerance of high substrate concentration of an esterase by directed evolution

A newly isolated enantioselective esterase from Pseudomonas fluorescens KCTC 1767, which is currently considered as a biocatalyst for the production of a commercially valuable (S)-ketoprofen, has revealed a low structural and thermal stability. In order to enhance the stability, directed evolution was attempted on this enantioselective esterase by successive steps of an error prone and staggered extension PCR. After the second round of evolution, the best mutant 6–52 with enhanced thermal stability was selected and analyzed. DNA sequence analyses of 6–52 revealed that the three amino acid residues (L120P, I208V, and T249A) were changed and the mutation L120P was presumed as a structurally important residue due to its presence in all positive variants. The purified mutant 6–52, when incubated at 50 and 55 °C for 2 h, remained its activity over 30 and 10%, respectively, whereas there were no detectable activities in wild-type enzyme. The analysis of 6–52 in the presence of 15% ethanol showed 1.8-fold increase in the activity, compared to that of wild-type enzyme. The K_m and V_max values of 6–52 were estimated to be slightly increased, leading to 1.2-fold-higher the catalytic efficacy k_cat/K_m than that of wild-type enzyme. Additionally, the mutant 6–52 was more resistant to high substrate concentrations than that of wild-type enzyme.     

Directed Evolution of E. coli Alkaline Phosphatase Towards Higher Catalytic Activity

Directed evolution was used to enhance the catalytic activity of E. coli alkaline phosphatase (EAP). Through two rounds of error-prone PCR and one round of DNA shuffling followed by a rapid, sensitive screening procedure, several improved variants were obtained. Their enzymatic kinetic properties, thermal stabilities and possible mechanism for the improvement were investigated. In 1.0 M Tris buffer, the specific activity of the most active EAP variant S2163 was 1500 units/mg protein, showing it to be 3.6 times more active than the D101S parent enzyme and ∼40 times more active than the wild-type EAP. At the same time, the K_m value of the S2163 variant decreased to 1491 μM from the 2384 μM of the D101S. As a result, the k_cat/K_m ratio of this variant showed a 5.8-fold enhancement over that of D101S parent enzyme. Three activating amino acid substitutions, K167R, G180S and S374C, which were located far away from the center of the catalytic pocket, were identified by sequencing the genes encoding evolved enzymes. Possible explanations for the improvement of activity were analyzed.     

SDS-subtilisin catalyzed synthesis of tetra-peptides containing multifunctional amino acid residues in ethanol

The role of acyl donor structure on the course of peptide bond formation catalyzed by SDS-subtilisin in ethanol was investigated. In the reaction Z---Ala---Ala---Leu---OR+H---Phe---pNA→Z---Ala---Ala---Leu---Phe---pNA, nearly quantitative product yields were observed after 2 h, regardless of whether an activated (R=CH₃, p-C₆H₅Cl) or non-activated (R=H) acyl donor was used. It was found that the enzyme can accept as acyl donors N-protected tri-peptides containing basic or acidic amino acid residues in the P₁-position. Tetra-peptides of general formula Z---Ala---Ala---P₁---P₁′---pNA, where P₁=Glu, Asp, Lys, Arg or His and P₁′=Phe, Arg or Glu have been obtained in good yield.     

Kinetic characterization of sulphur-containing excitatory amino acid uptake in primary cultures of neurons and astrocytes

The uptake of the neuroactive sulphur amino acids -cysteine sulphinate, -cysteate, -homocysteine sulphinate and -homocysteate was investigated in astrocytes cultured from the prefrontal cortex; in neurons, cultured from cerebral cortex; and, in granule cells, cultured from cerebellum. It was shown that each amino acid acted as a substrate for a plasma membrane transporter in both neurons and astrocytes. Astrocytes and neurons exhibited a high-affinity uptake for -cysteine sulphinate and -cysteate with K_m values ranging from 14–100 μM, and a low-affinity uptake for -homocysteine sulphinate and -homocysteate, with K_m values ranging from 225–1210 μM. The uptake of all transmitter candidates studied was partially sodium-dependent. This sodium-dependency was most evident at low (< 100 μM) concentrations of each substrate. The apparent uptake measured in the absence of sodium was included as a component in corrections made for non-saturable influx. With the exception of -cysteine sulphinate, uptake of each sulphur amino acid was greatest in astrocytes, with V_max values ranging between 15–32 nmol min⁻¹ mg⁻¹ cell protein. Moreover, the uptake of each sulphur amino acid in cerebellar granule cells (V_max values ranging between 10–25 nmol min⁻¹ mg⁻¹ cell protein) was consistently greater than that in cerebral cortex neurons (V_max values ranging between 1.5–6 nmol min⁻¹ mg⁻¹ cell protein).