Design, synthesis and catalytic activity of a serine protease synthetic model

Summary The design, synthesis and catalytic properties of a cyclic branched peptide carrier that possesses the catalytic triad residues of the serine proteases is reported. The synthesis of the peptide model was totally completed on solid support using three different orthogonal amino protecting groups. Hydrolytic activity measurements against Suc-Ala-Ala-Ala-pNA substrate showed that it is hydrolysed by the peptide model to a small extent. Despite this small hydrolytic activity, it is the first time, to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound. Contrary, this enzyme model peptide showed considerable activity against the Boc-Ala-pNP substrate (k _cat =0.414 min⁻¹ andK _m =0.228 mm). These results suggest that the designed carrier brings in appropriate contact the catalytic triad residues (Ser, His, Asp) resulting in the obtained hydrolytic activity.     

Helix-loop-helix peptide foldamers and their use in the construction of hydrolase mimetics

De novo designed helix-loop-helix peptide foldamers containing cis-2-aminocyclopentanecarboxylic acid residues were evaluated for their conformational stability and possible use in enzyme mimetic development. The correlation between hydrogen bond network size and conformational stability was demonstrated through CD and NMR spectroscopies. Molecules incorporating a Cys/His/Glu triad exhibited enzyme-like hydrolytic activity.     

An apoptosis-inducing serine protease secreted by the entomopathogenic nematode Steinernema carpocapsae

Steinernema carpocapsae is an insect parasitic nematode able to parasitise and kill the host within 48 h. Secreted products (ESP) of the parasitic stage of a virulent strain contain higher amounts of proteolytic activity than a low virulence strain, suggesting proteases are involved in virulence. From the ESP we purified a protein (Sc-SP-3) with a M_r of 30 kDa and a pI of 7 that cleaved the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA and was inhibited by phenylmethanesulfonyl fluoride, benzamidine and chymostatin, thus indicating that it belongs to the chymotrypsin-like serine protease family. Sc-SP-3 has a V_max of 0.3 mM min⁻¹ ml⁻¹ and K_m of 6.6 × 10⁻⁴ M, with maximum activity at pH 8 and 40 °C. The full-length cDNA was obtained using degenerate oligonucleotides for serine proteases. This open reading frame encodes a preproprotein containing a putative signal peptide composed of 16 amino acid residues, a prodomain of 40 residues and a mature protease domain of 261 residues, including the catalytic triad His/Asp/Ser characteristic of trypsin-like serine proteases. The N-terminal sequence and the peptide masses fingerprint obtained by MALDI-TOF–MS for the purified protein matched the cDNA. Gene expression analysis by quantitative real-time-PCR showed that this gene is expressed only during the parasitic stage and that pre-invasive nematodes inside the mid-gut expressed higher amounts of Sc-SP-3 than those that already enter the haemocoel. Sc-SP-3 caused histolysis in the insect mid-gut. In vitro assays demonstrated that Sc-SP-3 digested extracellular proteins and induced apoptosis in Sf9 insect cells, thus suggesting Sc-SP-3 is a multifunctional chymotrypsin-like protease involved in pathogenesis.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Coupling of proteolysis to ATP hydrolysis uponEscherichia coli lon protease functioning: I. kinetic aspects of ATP hydrolysis

Some aspects of theEscherichia coli Lon protease ATPase function were studied around the optimum pH value. It was revealed that in the absence of the protein substrate the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg²⁺ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)²⁻ inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg²⁺ ions is caused by the formation of the “ADP-Mg-form” of ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)²⁻ complex to the enzyme, by an elimination of the inhibiting action of free Mg²⁺ ions without altering the efficiency of catalysis of ATP hydrolysis (based on thek _cat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)⁻ complex (without changing theK _i value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.     

Computational,pulse-radiolytic,and structural investigations of lysine-136 and its role in the electrostatic triad of human C u,Z n superoxide dismutase

Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote electrostatic steering of the superoxide substrate to the active site Cu ion, resulting in dismutation of superoxide to oxygen and hydrogen peroxide. Lys-136, along with the adjacent residues Glu-132 and Glu-133, forms a proposed electrostatic triad contributing to substrate recognition. Human Cu,Zn SODs with single-site replacements of Lys-136 by Arg, Ala, Gln, or Glu or with a triple-site substitution (Glu-132 and Glu-133 to Gln and Lys-136 to Ala) were made to test hypotheses regarding contributions of these residues to Cu,Zn SOD activity. The structural effects of these mutations were modeled computationally and validated by the X-ray crystallographic structure determination of Cu,Zn SOD having the Lys-136-to-Glu replacement. Brownian dynamics simulations and multiple-site titration calculations predicted mutant reaction rates as well as ionic strength and pH effects measured by pulse-radiolytic experiments. Lys-136-to-Glu charge reversal decreased dismutation activity 50% from 2.2 × 10⁹ to 1.2 × 10⁹ M⁻¹ s⁻¹ due to repulsion of negatively charged superoxide, whereas charge-neutralizing substitutions (Lys-136 to Gln or Ala) had a less dramatic influence. In contrast, the triple-mutant Cu,Zn SOD (all three charges in the electrostatic triad neutralized) surprisingly doubled the reaction rate compared with wild-type enzyme but introduced phosphate inhibition. Computational and experimental reaction rates decreased with increasing ionic strength in all of the Lys-136 mutants, with charge reversal having a more pronounced effect than charge neutralization, implying that local electrostatic effects still govern the dismutation rates. Multiple-site titration analysis showed that deprotonation events throughout the enzyme are likely responsible for the gradual decrease in SOD activity above pH 9.5 and predicted a pK_a value of 11.7 for Lys-136. Overall, Lys-136 and Glu-132 make comparable contributions to substrate recognition but are less critical to enzyme function than Arg-143, which is both mechanistically and electrostatically essential. Thus, the sequence-conserved residues of this electrostatic triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while simultaneously providing specificity by selecting against binding by other anions. Proteins 29:103–112, 1997. © 1997 Wiley-Liss, Inc.     

Point mutation of (+)-germacrene A synthase from <Emphasis Type="Italic">Ixeris dentata</Emphasis>

Sesquiterpene cyclases catalyze the conversion of common precursor, farnesyl pyrophosphate, into various terpene backbones. X-ray crystallography of tobacco epi-aristolochene synthase has previously proposed a cyclization mechanism wherein the allylic carbocation intermediate is stabilized by the main chain carbonyl oxygens of three consecutive threonine residues. Alignment of amino acid sequences of plant terpene cyclases shows that the first position of the triad is almost invariably threonine or serine. To probe the carbocation-stabilizing role, the amino acid residues of the ₄₃₃TSA₄₃₅ triad in (+)-germacrene A synthase from Ixeris dentata were altered by site-directed mutagenesis. Enzyme kinetic measurements of the mutants and GC/MS analysis of the enzyme reaction products indicate that mutations of the triad decreased enzyme catalysis rather than substrate binding but did not affect its structural rearrangement in the catalytic mechanism. This is the first report that the hydroxyl group of threonine at the first position of the triad is required for the cyclase activity.     

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep)

Aims: Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease‐catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent‐tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. Methods and Results: The tripeptide Ac‐Phe‐Gly‐Phe‐NH₂ was synthesized using Ac‐Phe‐OEt and Gly‐Phe‐NH₂ substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1·5 or 0·5 mol l^?1 NaCl. Purification and identification of the peptide product was achieved by RP‐HPLC and ESI‐MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. Conclusion: Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. Significance and Impact of Study: The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.     

Preparation and Characterization of a Truncated Caricain Lacking 41 Residues from the N-terminal

We purified an 18.8 kD protease from caricain solution. This protease was derived from caricain. It does not have the first 41 residues of the N-terminal sequence of caricain, and its N-terminal residue is Thr. Also, one of the disulfide bonds of caricain (cys²²–cys⁶³) was opened during the formation of the protease. We named this 18.8 kD protease caricain II. Caricain II has a wide pH range, and it is more sensitive to temperature changes than caricain. The proteolytic activity of caricain II is twice as much as that of caricain using casein as a substrate. However, caricain II has a low hydrolytic activity with N-benzoyl-l-arginine ethyl ester (BAEE) that is one of the special substrates of caricain. Our results indicate that caricain II is remarkably different from caricain and it can provide an improvement over caricain on the proteolytic activity.     

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C₈-acyl carrier protein (C₈-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4′-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C₈-CoA. Furthermore, our ¹H saturation transfer difference and ³¹P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved ⁷¹RGG⁷³ loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.     

Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8

Proteases are the hydrolytic enzymes which hydrolyzes peptide bond between proteins with paramount applications in pharmaceutical and industrial sector. Therefore production of proteases with efficient characteristics of biotechnological interest from novel strain is significant. Hence, in this study, an alkaline serine protease produced by Bacillus cereus strain S8 (MTCC NO 11901) was purified and characterized. The alkaline protease was purified by ammonium sulfate precipitation (50%), ion exchange (DEAE-Cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. As a result of this purification, a protein with specific activity of 300U/mg protein was obtained with purification fold 17.04 and recovery percentage of 34.6%. The molecular weight of the purified protease was determined using SDS-PAGE under non-reducing (71?kDa) and reducing conditions (35?kDa and 22?kDa). Zymogram analysis revealed that proteolytic activity was only associated with 22?kDa. These results indicate that existence of the enzyme as dimer in its native state. The molecular weight of the protease (22?kDa) was also determined by gel filtration (Sephadex G-200) chromatography and it was calculated as 21.8?kDa. The optimum activity of the protease was observed at pH 10.0 and temperature 70?°C with great stability towards pH and temperature with casein as a specific substrate. The enzyme was completely inhibited by PMSF and TLCK indicating that it is a serine protease of trypsin type. The enzyme exhibits a great stability towards organic solvents, oxidizing and bleaching agents and it is negatively influenced by Li²⁺ and Co²⁺ metal ions. The purified protein was further characterized by Matrix Assisted Laser Desorption Ionization/Mass Spectroscopy (MALDI/MS) analysis which reveals that total number of amino acids is 208 with isoelectric point 9.52.     

Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.     

Bovine pancreatic elastase II cleaves Gln-Ile bond

A peptidase (GICP) that cleaves the Gln-Ile bond of a peptide Gly-Ile-Asp-Val-Gln-Ile-Tyr(T-1), a sequence in phenylalanine oxidase, was purified from bovine pancreas. The purified enzyme had an Mr of approximately 29,000, as determined by SDS-PAGE, and its N-terminal sequence was identical to that of bovine pancreatic elastase II. The enzyme released Gly-Ile-Asp-Val-Gln and Ile-Tyr from T-1 (Km = 8.3 M k_cat = 2.1 s^–1) and the catalytic efficiency (2.6 × 10⁵ M^–1s^–1) was comparable to those of elastase II from porcine pancreas and rat mesenteric arterial bed perfusate. The P₁ site specificity of GICP toward oxidized insulin A and B chains suggested that major cleavage sites were the peptide bond at the C-terminal side of Gln, Leu, His, and Tyr residues.     

Eryngase: a Pleurotus eryngii aminopeptidase exhibiting peptide bond formation activity

An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately 79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic l-aminoacyl-pNAs and exhibited little activity toward d-Ala-pNA and d-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors. The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore, eryngase has produced opioid dipeptides––l-kyotorphin (l-Tyr-l-Arg) and d-kyotorphin (l-Tyr-d-Arg)––using l-Tyr-NH₂ and d- and l-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%.     

Demonstration and characterization of hippocampal cholinergic neurostimulating peptide (HCNP) processing enzyme activity in rat hippocampus

Hippocampal cholinergic neurostimulating peptide (HCNP) stimulates cholinergic activity of cultured medial septal nuclei explants. It consists of eleven amino acids that are located at the N-terminal region of its precursor protein. This report concerns the demonstration and characterization of an HCNP processing enzyme that cleaves the bioactive undecapeptide from the precursor. The enzyme was purified from the hippocampus of young Wistar rats. A synthetic deacetylated peptide (peptide_1–26) consisting of the N-terminal 26 amino acids of the HCNP precursor protein served as substrate. The product of the enzyme reaction was identified and quantitated by HPLC using deacetylated HCNP as standard. The amount of undecapeptide generated was directly proportional to the time of incubation of the enzyme reaction mixture. From molecular sieving chromatography it was estimated that the molecular mass of the enzyme is close to 68 kDa. The HCNP processing enzyme has a pH optimum of 6.0 and a K_m of 0.50 mM for peptide_1–26. Preincubation at 56°C causes rapid inactivation of the HCNP processing activity. Enzyme activity is enhanced by EDTA and 1,4-dithiothreitol, and inhibited by antipain, chymostatin and E-64. These findings suggest that the enzyme probably has a thiol group in its active site. This novel enzyme of the hippocampus may represent a valuable tool for further studies on the general protein metabolism in the central nervous system, as well as for elucidating the neurochemical aspects of neurodegenerative disorders.     

Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C–C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 Å resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical α/β-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C–C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr¹²¹, Tyr²²⁹, and Asn¹³², which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.