Protease-catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid-phase peptide synthesis with enzymatic methods.

The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in the site-specific amino acid moiety from the acyl residue to the ester-leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid-phase peptide synthesis with substrate mimetic-mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4-guanidinophenyl-, phenyl- and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N(alpha)-protected peptide esters as acyl donors in trypsin-, alpha-chymotrypsin- and V8 protease-catalyzed fragment condensations.     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Non-coded amino acids as acyl donor substrates for peptide bond formation catalyzed by thermoase in toluene

The peptide bond formation of N-protected non-coded amino acids having different structures as acyl donor substrates that is catalyzed by thermoase in organic media was investigated. In these reactions, N-protected l--non-coded amino acids, including l-Orn, l-Cit, -aminobutyric acid (l--Abu) and phenylalanine homologues, were used as the acyl donors and phenylalanine derivatives were used as the acyl acceptors. This kind of enzymatic reactions cannot be carried out in an aqueous buffer due to the rigid specificity of proteases to coded amino acids in water. The results demonstrated that the substrate specificity of proteases could be broadened in organic solvents. In addition, the factors that influenced these protease-catalyzed reactions, including structures of the substrates, water content and the bases used, were systematically studied. Our work provided important evidence for broadening the application of protease in organic synthesis.     

Trypsin-specific acyl-4-guanidinophenyl esters for alpha-chymotrypsin-catalysed reactions computational predictions, hydrolyses, and peptide bond formation.

The function of acyl-4-guanidinophenyl esters as substrate mimetics for the serine protease alpha-chymotrypsin was investigated by protein-ligand docking, hydrolysis, and acyl transfer experiments. On the basis of protein-ligand docking studies, the binding and hydrolysis properties of these artificial substrates were estimated. The predictions of the rational approach were confirmed by steady-state hydrolysis studies on 4-guanidinophenyl esters derived from coded amino acids (which alpha-chymotrypsin is not specific for), noncoded amino acids, and even simple carboxylic acid moieties. Enzymatic peptide syntheses qualify these esters as suitable acyl donors for the coupling of acyl components far from the natural enzyme specificity, thus considerably expanding the synthetic utility of alpha-chymotrypsin.     

Protease-catalyzed hydrolysis of substrate mimetics (inverse substrates): A new approach reveals a new mechanism.

Contrary to common protease substrates, the hydrolysis of 4-guanidinophenyl esters of the Boc-Xaa-OGp type by trypsin and trypsin-like proteases performs easily and independently of the structure and chirality of the acyl moiety. The hydrolysis of this new class of substrate mimetics, previously called inverse substrates, is enabled by the highly specific leaving group. However, the mechanism cannot be explained on the basis of the conventional binding model which defines the interactions between the protease and its substrate. Hydrolysis and aminolysis kinetics, protein-ligand docking, and molecular dynamics studies have been carried out in order to get insight into the catalytic mechanism which holds for these substrate mimetics. The experimental and theoretical results obtained for the serine protease trypsin suggest a novel extended kinetic model. It explains the hydrolysis of these types of protease substrates and accounts for the structural consequences for their aminolysis.     

Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by 18O isotope exchange

The peptidolytic reaction of HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2, that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55gag and Pr160gag-pol. The values for the kinetic parameters V/KEt and V/Et were 0.16-7.5 mM-1 s-1 and 0.24-29 s-1, respectively, at pH 6.0, 0.2 M NaCl, and 37 degrees C. By use of a variety of inorganic salts, it was concluded that the peptidolytic reaction is nonspecifically activated by increasing ionic strength. V/K increased in an apparently parabolic fashion with increasing ionic strength, while V was either increased or decreased slightly. From product inhibition studies, the kinetic mechanism of the protease is either random or ordered uni-bi, depending on the substrate studied. The reverse reaction or a partial reverse reaction (as measured by isotope exchange of the carboxylic product into substrate) was negligible for most of the oligopeptide substrates, but the enzyme catalyzed the formation of Ac-Ser-Gln-Asn-Tyr-Phe-Leu-Asp-Gly-NH2 from the products Ac-Ser-Gln-Asn-Tyr and Phe-Leu-Asp-Gly-NH2. The protease-catalyzed exchange of an atom of 18O from H2 18O into the re-formed substrates occurred at a rate which was 0.01-0.12 times that of the forward peptidolytic reaction. The results of these studies are in accord with the formation of a kinetically competent enzyme-bound amide hydrate intermediate, the collapse of which is the rate-limiting chemical step in the reaction pathway.     

An enigmatic peptide ligation reaction: protease-catalyzed oligomerization of a native protein segment in neat aqueous solution

We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free alpha-carboxyl and alpha-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 degrees C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases-slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase."     

Molecular insights into substrate specificity of prostate specific antigen through structural modeling

Prostate Specific Antigen's (PSA) role as a biomarker for prostate cancer is well established but the physiological role of its serine protease activity in the pathobiology of normal prostate and prostate carcinogenesis remains largely unknown. In light of recent studies that implicate PSA's enzymatic activity in the initiation and/or progression of prostate cancer, we performed a molecular modeling study of substrate binding at the catalytic site of PSA wherein a PSA‐selective substrate (HSSKLQ) was docked in an acyl‐enzyme conformation to a three‐dimensional homology model of PSA. Additionally, virtual positional scanning studies were conducted to gain mechanistic insights into substrate recognition of PSA. Subsequently, 13 novel peptide substrates of 6‐aa length and four peptide substrates with varying length were synthesized and assayed for PSA hydrolysis to evaluate the experimental validity of docking insights. Additionally, six novel aldehyde‐containing transition state analog inhibitors were synthesized and tested for their inhibitory potencies. The experimental data on the hydrolysis rates of the newly synthesized substrates and inhibitory potencies of the aldehyde peptides agreed with the docking predictions, providing validation of the docking methodology and demonstrating its utility towards the design of substrate‐mimetic inhibitors that can be used to explore PSA's role in the pathobiology of prostate cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Peptide bond formation mediated by substrate mimetics. Structure-guidedoptimization of trypsin for synthesis.

Substrate mimetics are excellent tools for protease-mediated peptide synthesis that enable the coupling of peptides independently of the primary specificity of the enzyme without undesired cleavages of the newly formed peptide bonds. However, the synthetic utility of this beneficial approach is limited to reactions with nonspecific amino-acid-containing peptides while the coupling of specific ones leads to unwanted cleavages due to the native proteolytic activity of the biocatalyst. This paper reports on the use of site-directed mutagenesis to design trypsin variants with decreased cleavage activity. Starting from the variant D189S, which is known for its low proteolytic potential, Ser189 and Ser190 were exchanged for Ala to further repress the inherent amidase activity of trypsin D189S. The effect of mutations was analysed by model synthesis reactions using specific amino-acid-containing peptides and substrate mimetics as the reactants. Finally, computer-assisted protein-ligand docking studies were performed to get closer insight into the molecular basis of the experimental results.     

A direct spectrophotometric assay for peptide deformylase.

A direct UV-VIS spectrophotometric assay has been developed for peptide deformylase. This assay employs a novel class of peptide mimetics as deformylase substrates which, upon enzymatic removal of the N-terminal formyl group, rapidly release free thiols. The released thiols are quantitated using Ellman's reagent. A variety of peptide analogues that contain beta-thiaphenylalanine or beta-thiamethionine as the N-terminal residue were synthesized and found to be excellent substrates of the peptide deformylase from Escherichia coli (k(cat)/K(M) = 6.9 x 10(5) M(-1) s(-1) for the most reactive substrate). The deformylase reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion. The versatility of the assay has been demonstrated by its application to kinetic characterization of the deformylase, pH profile studies, and enzyme inhibition assays. The assay can also be performed in an end-point fashion. The results demonstrate that this assay is a simple, highly sensitive, and rapid method to study kinetic properties of deformylases without the use of any coupling enzymes.     

A novel and efficient enzymatic method for the production of peptides from unprotected starting materials

We report herein the development of a novel and efficient enzymatic method for the production of oligopeptides. This newly discovered method is a simple, cost-effective process, using unprotected amino acids as substrates in an aqueous solution and producing peptides in high yield. The target of our initial screen was l-alanyl-L-glutamine, a dipeptide of significant industrial interest by virtue of its widespread use in infusion therapy. By means of the screening of microorganisms that can catalyze the peptide-forming reaction producing l-alanyl-L-glutamine from L-alanine methylester (acyl donor) and L-glutamine (nucleophile), we discovered that Empedobacter brevis ATCC 14234 produced l-alanyl-L-glutamine most efficiently. The newly found enzyme purified from E. brevis ATCC 14234 facilitates significantly high production yields of l-alanyl-L-glutamine from L-alanine methylester and L-glutamine in an aqueous solution--more than 80% yield based on L-alanine methylester. In addition, this enzyme has wide substrate specificity--both for acyl donors and nucleophiles--and can catalyze peptide-forming reactions not only to produce various dipeptides from the corresponding amino acid esters and amino acids but also to produce various oligopeptides from the corresponding amino acid esters and peptides.     

Synthesis of dermorphin-(1-4) derivatives catalyzed by proteases in organic solvents.

In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing D-amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) with potent analgesic activity. The N-terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin-(1-4) derivatives, Boc-Tyr-D-Ala-Phe-Gly-N(2)H(2)Ph and Boc-Tyr-D-Ala-Phe-Gly-NH(2), which contained a d-amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin-(1-4) derivatives could be catalyzed by subtilisin with Boc-Tyr-D-Ala-OCH(2)CF(3) as an acyl donor substrate in AcOEt. The synthesis of dermorphin-(1-2) derivative Boc-Tyr-D-Ala-N(2)H(2)Ph was catalyzed by alpha-chymotrypsin in different organic solvents and D-Ala-N(2)H(2)Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.     

Genome scale prediction of substrate specificity for acyl adenylate superfamily of enzymes based on active site residue profiles

Background  

Enzymes belonging to acyl:CoA synthetase (ACS) superfamily activate wide variety of substrates and play major role in increasing the structural and functional diversity of various secondary metabolites in microbes and plants. However, due to the large sequence divergence within the superfamily, it is difficult to predict their substrate preference by annotation transfer from the closest homolog. Therefore, a large number of ACS sequences present in public databases lack any functional annotation at the level of substrate specificity. Recently, several examples have been reported where the enzymes showing high sequence similarity to luciferases or coumarate:CoA ligases have been surprisingly found to activate fatty acyl substrates in experimental studies. In this work, we have investigated the relationship between the substrate specificity of ACS and their sequence/structural features, and developed a novel computational protocol for in silico assignment of substrate preference.     

An apparatus for continuous analysis of protease-catalyzed acyl transfer reactions

An apparatus that allows continuous analysis of protease-catalyzed acyl transfer reactions is described. Hydrolysis reaction is assayed using automatic titration. A continuous determination of amino group concentration by reaction with o-phthalaldehyde gives the rate of peptide bond formation. The apparatus allows the determination of the partition constant for the nucleophile at various nucleophile concentrations from one run.     

Characterization of rat liver bile acid acyl glucuronosyltransferase

Recent studies have suggested that bile acid acyl glucuronides form covalently bound protein adducts which may cause hypersensitivity reactions and increased morbidity in patients. Although the preferential biosynthesis of the acyl glucuronides has been known, the characterization of hepatic bile acid acyl glucuronosyltransferase has not yet been clearly elucidated. We have investigated the substrate specificity of the hepatic bile acid acyl glucuronosyltransferase using five common bile acids as substrates. The glucuronidation rate was dependent on the number of the hydroxy group on the steroid nucleus and mono-hydroxylated lithocholic acid, the more lipophilic common bile acid, was most effectively metabolized into its acyl glucuronide. The tri-hydroxylated cholic acid, the more water-soluble common bile acid, barely transformed into its glucuronide. Results showed decreasing of the initial velocity of the acyl glucuronidation with increasing of the concentration of substrate, lithocholic acid, owing to the substrate inhibition of the hepatic bile acid acyl glucuronosyltransferase. The substrate analogues, glycine and taurine conjugated bile acids, which exist in the body fluids in high concentrations, also inhibited the enzyme's activity. In addition, enzymatic reaction products, bile acid acyl glucuronides, also inhibited the activity. These inhibitory mechanisms may be responsible for the low concentration of bile acid acyl glucuronides in urine and may be an important detoxification system in the body.     

Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate.

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein. The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate, trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general "U-shaped" conformation, with the trans double bond positioned directly adjacent to the nicotinamide ring of NAD+. The side chain of Tyr158 directly interacts with the thioester carbonyl oxygen of the C16 fatty acyl substrate and therefore could help stabilize the enolate intermediate, proposed to form during substrate catalysis. Hydrophobic residues, primarily from the substrate binding loop (residues 196-219), engulf the fatty acyl chain portion of the substrate. The substrate binding loop of InhA is longer than that of other enoyl-ACP reductases and creates a deeper substrate binding crevice, consistent with the ability of InhA to recognize longer chain fatty acyl substrates.     

A prokaryotic acyl-CoA reductase performing reduction of fatty acyl-CoA to fatty alcohol

The reduction of acyl-CoA or acyl-ACP to fatty alcohol occurs via a fatty aldehyde intermediate. In prokaryotes this reaction is thought to be performed by separate enzymes for each reduction step while in eukaryotes these reactions are performed by a single enzyme without the release of the intermediate fatty aldehyde. However, here we report that a purified fatty acyl reductase from Marinobacter aquaeolei VT8, evolutionarily related to the fatty acyl reductases in eukaryotes, catalysed both reduction steps. Thus, there are at least two pathways existing among prokaryotes for the reduction of activated acyl substrates to fatty alcohol. The Marinobacter fatty acyl reductase studied has a wide substrate range in comparison to what can be found among enzymes so far studied in eukaryotes.     

Structure and function of the RedJ protein, a thioesterase from the prodiginine biosynthetic pathway in Streptomyces coelicolor

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a β-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.     

Protease-catalyzed synthesis of a precursor dipeptide, Z-Asp-Val-NH2 of thymopentin, in organic solvents

The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide, Z-Asp-Val-NH(2) of thymopentin (TP-5), in organic solvents was studied. Z-Asp-OMe and Val-NH(2) were used as the acyl donor and the nucleophile, respectively. An industrial alkaline protease alcalase was used to catalyze the synthesis of the target dipeptide in water-organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-NH(2). The optimum conditions using alcalase as the catalyst are pH 10.0, 35 degrees C, in acetonitrile/Na(2)CO(3)-NaHCO(3) buffer system (9:1, V/V), reaction time 5 h, with a yield of 63%. The dipeptide product was confirmed by LC- MS.