Kinetics and phospholipid specificity of apolipoprotein N-acyltransferase

The enzyme apolipoprotein N-acyltransferase (Lnt) is an integral membrane protein that catalyzes the last step in the post-translational modification of bacterial lipoproteins. Lnt undergoes covalent modification in the presence of phospholipids resulting in a thioester acyl-enzyme intermediate. It then transfers the acyl chain to the α-amino group of the N-terminal diacylglyceryl-modified cysteine of apolipoprotein, leading to the formation of mature triacylated lipoprotein. To gain insight into the catalytic mechanism of this two-step reaction, we overproduced and purified the enzyme of Escherichia coli and studied its N-acyltransferase activity using a novel in vitro assay. The purified enzyme was fully active, as judged by its ability to form a stable thioester acyl-enzyme intermediate and N-acylate the apo-form of the murein lipoprotein Lpp in vitro. Incorporation of [(3)H]palmitate and mass spectrometry analysis demonstrated that Lnt recognized the synthetic diacylglyceryl-modified lipopeptide FSL-1 as a substrate in a mixed micelle assay. Kinetics of Lnt using phosphatidylethanolamine as an acyl donor and FSL-1 as a substrate were consistent with a ping-pong type mechanism, demonstrating slow acyl-enzyme intermediate formation and rapid N-acyl transfer to the apolipopeptide in vitro. In contrast to earlier in vitro observations, the N-acyltransferase activity was strongly affected by the phospholipid headgroup and acyl chain composition.     

Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis

Atomic resolution (相似文献   

Acyl-enzyme complexes between tissue-type plasminogen activator and neuroserpin are short-lived in vitro

The serine protease tissue-type plasminogen activator (t-PA) initiates the fibrinolytic protease cascade and plays a significant role in motor learning, memory, and neuronal cell death induced by excitotoxin and ischemia. In the fibrinolytic system, the serpin PAI-1 negatively regulates the enzymatic activity of both single-chain and two-chain t-PA (sct-PA and tct-PA). In the central nervous system, neuroserpin (NSP) is a serpin thought to regulate t-PA enzymatic activity. We report that although both sct-PA and tct-PA rapidly form acyl-enzyme complexes with NSP in vitro, the interactions are short-lived, rapidly progressing to complete cleavage of NSP and regeneration of fully active enzyme. All NSP molecules appear to transit through the detectable acyl-enzyme intermediate and progress to completion of cleavage; no subpopulation that functions as a pure substrate was detected. Likewise, all molecules were reactive, with no evidence of a latent subpopulation. The interactions between NSP and t-PA were distinct from those between plasmin and NSP, wherein the same peptide bond was cleaved but there was no evidence of a detectable plasmin-NSP acyl-enzyme complex. The interactions between t-PA and NSP contrast with the formation of long-lived, physiologically irreversible acyl-enzyme complexes between t-PA and PAI-1, suggesting that the physiologic effect of t-PA-NSP interactions may be more complex than previously thought.     

Peptide synthesis catalyzed by proteases. Analysis of a kinetic model for enzymes with acyl-enzyme mechanism of action

The kinetics of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids or peptides (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate, was analysed. A kinetic model assumes enzymatic hydrolysis of the formed peptide (P), so the kinetic curve for P has a maximum (denoted as pmax). Particular attention was given to the analysis of the effects of the initial concentrations and kinetic constants on pmax. Computer analysis demonstrated that at a given ratio of initial S and N concentrations pmax is affected only by the ratio of the second order rate constants for enzymatic hydrolysis of S and P (alpha) and the ratio of rate constants for an attack of the acyl-enzyme intermediate by nucleophile and water (beta). These conclusions apply regardless of the existence of enzyme forms other than a free enzyme and an acyl-enzyme intermediate. Thus, the kinetically controlled maximum yield of peptide (pmax) can be calculated a priori from the values of alpha and beta which can be readily evaluated from the reference data. Simple explicit expressions were obtained, allowing fairly accurate prediction of pmax for a broad spectrum of S and N initial concentrations.     

A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein

We report here the use of TEV protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins. Bacterial expression was utilized to produce two fusion proteins, GPRT-C37-H6 and His-tagged interleukin-2 (amino acids 6-133), which when cleaved by the tobacco etch virus NIa protease (TEV protease) to generate HIV entry inhibitor peptide C37-H6 and a truncated version of the cytokine interleukin-2, both containing N-terminal cysteines. The N-terminal cysteine containing C37-H6 and truncated interleukin-2 were then joined to a synthetic glycopeptide thioester utilizing native chemical ligation under nondenaturing and denaturing conditions, respectively. The ligations of the glycopeptide to the C37-H6 peptide and the truncated interleukin-2 protein both proceeded in high yield, though the size, and physical properties of the two polypeptides differ greatly.     

HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis.

Hemolysin, a toxic protein produced by pathogenic Escherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria. HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin. Acyl-acyl carrier protein is the essential acyl donor. The acyltransferase reaction progresses through formation of a binary complex between acyl-ACP and HlyC to a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. The homologous acyltransferases of the family have a number of conserved amino acid residues that may be catalytically important. Experiments to illuminate the reaction mechanism were done. The formation of an acyl-enzyme intermediate suggested that the reaction likely proceeded through two partial reactions. The reversibility of the first partial reaction was shown by using separately subcloned, purified, and expressed substrates and enzyme. The effects of single site-directed mutations of conserved residues of HlyC on different portions of reaction progress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined. Mutations of His23, the only residue essential for activity, formed normal binary complexes but were unable to form acyl-HlyC. The same was seen with S20A, a mutant with greatly impaired activity. Mutation of two conserved tyrosines separately to glycines results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-type.     

Increasing solubility of proteins and peptides by site-specific modification with betaine

Proteins and peptides with low solubility and which aggregate are often encountered in biochemical studies and in pharmaceutical applications of polypeptides. Here, we report a new strategy to improve solubility and prevent aggregation of polypeptides using site-specific modification with the small molecule betaine, which contains a quaternary ammonium moiety. Betaine was site-selectively attached to the N-termini of two aggregation-prone polypeptide models, the bacterial enzyme xanthine-guanine phosphoribosyltransferase (CG-GPRT) and the HIV entry inhibitor peptide CG-T20, utilizing native chemical ligation. N-terminal cysteines for the betaine ligation reactions were generated from His-tagged fusion proteins using TEV protease cleavage. Ligation of the betaine thioester (1) to the N-terminal cysteine-containing polypeptide models proceeded in high yield, though denaturing conditions were required for CG-T20 due to the hydrophobic nature of this peptide. CD spectroscopy and GPRT activity assays indicate that the betaine modification of CG-GPRT and CG-T20 does not significantly affect structure or activity of the polypeptides. Solubility and turbidity measurements of betaine-modified and unmodified polypeptides demonstrate that betaine modification can greatly increase solubility. Finally, it is shown that betaine-modified CG-T20 acts as an inhibitor of the aggregation of unmodified CG-T20.     

Inhibitors can activate proteases to catalyze the synthesis and hydrolysis of peptides

Competitive inhibitors can activate proteases (papain, trypsin, and cathepsin S) to catalyze the synthesis of peptide bonds and accelerate the hydrolysis of poor substrates (from 1 to 99%). Reaction mixtures contained intermediate molecules that were formed by the coupling of the inhibitor with the poor substrate. This and other findings suggest the following chain of events. Part of the binding energy of formation of the enzyme-inhibitor complex was used to activate the inhibitor, i.e., to form acyl-enzyme species with a high-energy bond (e.g., a thioester bond in the case of papain) required for coupling the inhibitor with the substrate to form the intermediate molecule. The latter was subjected to successive reactions which led to a stepwise degradation of the substrate, as well as to the regeneration of the inhibitor. One mole of the inhibitor could catalyze rapid hydrolysis of at least 53 mol of substrate. The intermediate molecules were the species undergoing rapid hydrolysis. Therefore, 1 mol of inhibitor was involved in the synthesis of 53 mol of intermediate molecules; i.e., the inhibitor functioned as a cofactor that catalyzed the synthesis of peptides. Thus, the binding energy of formation of the enzyme-inhibitor complex can be utilized to catalyze the synthesis of peptide bonds in the absence of an exogenous energy source (e.g., ATP).     

Isopeptide ligation catalyzed by quintessential sortase A: mechanistic cues from cyclic and branched oligomers of indolicidin

The housekeeping transpeptidase sortase A (SrtA) from Staphyloccocus aureus catalyzes the covalent anchoring of surface proteins to the cell wall by linking the threonyl carboxylate of the LPXTG recognition motif to the amino group of the pentaglycine cross-bridge of the peptidoglycan. SrtA-catalyzed ligation of an LPXTG containing polypeptide with an aminoglycine-terminated moiety occurs efficiently in vitro and has inspired the use of this enzyme as a synthetic tool in biological chemistry. Here we demonstrate the propensity of SrtA to catalyze "isopeptide" ligation. Using model peptide sequences, we show that SrtA can transfer LPXTG peptide substrates to the ε-amine of specific Lys residues and form cyclized and/or a gamut of branched oligomers. Our results provide insights about principles governing isopeptide ligation reactions catalyzed by SrtA and suggest that although cyclization is guided by distance relationship between Lys (ε-amine) and Thr (α-carboxyl) residues, facile branched oligomerization requires the presence of a stable and long-lived acyl-enzyme intermediate.     

An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase

Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys¹⁶¹ residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis.     

The thioesterase domain from the pimaricin and erythromycin biosynthetic pathways can catalyze hydrolysis of simple thioester substrates

The recombinant polyketide synthase thioesterase domains from the pimaricin and 6-deoxyerythronolide B biosynthetic pathways catalyze hydrolysis of a number of simple N-acetylcysteamine thioester derivatives. This study demonstrates that thioesterases are not highly substrate selective in formation of the acyl-enzyme intermediate, in contrast to non-ribosomal peptide synthase thioesterase domains that show very high specificity for substrate loading. This observation has important implications for the engineering of biosynthetic pathways to produce polyketide products.     

Kinetic specificity in papain-catalysed hydrolyses

The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme-substrate complex. Models for the enzyme-substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme-substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme-substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.     

The effect of pepstatin A, an inhibitor of the pro-opiomelanocortin (POMC)-converting enzyme, on POMC processing in mouse intermediate pituitary

In our previous studies, we have purified a unique, paired basic residue-specific, prohormone-converting enzyme from pituitary intermediate lobe secretory vesicles. This enzyme, an aspartyl protease, was shown to cleave the intermediate lobe prohormone, pro-opiomelanocortin (POMC), to adrenocorticotropin, beta-endorphin and a 16 kDa NH2-terminal glycopeptide, in vitro [(1985) J. Biol. Chem. 260, 7194-7205]. To provide some evidence that this enzyme plays a role in prohormone conversion in the intact cell, the ability of pepstatin A, an aspartyl protease inhibitor, to block POMC processing in the mouse intermediate pituitary was investigated. By the use of a radioactive pulse-chase paradigm, [3H]POMC processing was found to be inhibited by 36.4% in pepstatin A-treated intermediate lobes. This result is consistent with the inactivation of pro-opiomelanocortin-converting enzyme by pepstatin A in the intact pituitary and further supports a role of this enzyme in POMC processing in vivo.     

Native chemical ligation derived method for recombinant peptide/protein C-terminal amidation

C-terminal amidation is often a requisite structural feature for peptide hormone bio-activity. We report a chemical amidation method that converts peptide/protein thioesters into their corresponding C-terminal amides. The peptide/protein thioester is treated with 1-(2,4-dimethoxyphenyl)-2-mercaptoethyl auxiliary (1b) in a native chemical ligation (NCL) reaction to form an intermediate, which upon removal of the auxiliary with TFA, yields the peptide/protein amide. We have demonstrated the general utility of the approach by successfully converting several synthetic peptide thioesters to peptide amides with high conversion rates. Preliminary results of converting a recombinant peptide thioester to its amide form are also reported.     

The ClpP N-terminus coordinates substrate access with protease active site reactivity

Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpPDeltaN exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a "gate" controlling substrate access to the active sites, (2) binding of ClpA opens this "gate", allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal "gate" stimulates acyl-enzyme hydrolysis.     

Cryoenzymology of staphylococcal beta-lactamase: trapping a serine-70-linked acyl-enzyme

Various cryosolvents were investigated for their suitability in cryoenzymological experiments with beta-lactamase from Staphylococcus aureus PC1. On the basis of the minimal effects on the catalytic and structural properties of the enzyme, ternary solvents containing ethylene glycol, methanol, and water were found most suitable. The interaction of beta-lactamase with a number of substrates was studied at subzero temperatures. In general, the reaction profiles were similar to those in aqueous solution at above-zero temperatures, with the exception of the slower rates. For cephalosporin substrates, such as PADAC, in which the 3'-substituent may leave to form a more stable form of the acyl-enzyme [Faraci, W., & Pratt, R. (1985) Biochemistry 24, 903-910], this intermediate could be readily stabilized at subzero temperatures. At -40 degrees C the slow rate of deacylation in the reaction with the chromophoric substrate 6 beta-[(furylacryloyl)amino]penicillanic acid permitted the acyl-enzyme to be stoichiometrically accumulated. This intermediate was then stabilized at low pH with trifluoroacetic acid. Isolation by centrifugal gel filtration, followed by pepsin digestion, gave a penicilloyl-labeled peptide which was isolated by HPLC. Subsequent trypsinolysis of this peptide gave a single labeled peptide, corresponding to the octapeptide surrounding the active-site serine, Ser-70.     

Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.     

The rate-determining step in pepsin-catalysed reactions, and evidence against an acyl-enzyme intermediate

To delineate further the pathway of pepsin-catalysed reactions, three types of experiments were performed: (a) the enzyme-catalysed hydrolysis of a number of di- and tri-peptide substrates was studied with a view to observing the rate-determining breakdown of a common intermediate; (b) the interaction of pepsin with several possible substrates for which ;burst' kinetics might be expected was investigated; (c) attempts were made to trap a possible acyl-enzyme intermediate with [(14)C]methanol in both a hydrolytic reaction (with N-acetyl-l-phenylalanyl-l-phenylalanylglycine) and in a ;virtual' reaction (with N-acetyl-l-phenylalanine) under conditions where extensive hydrolysis or (18)O exchange is known to occur. It is concluded that (i) intermediates in pepsin-catalysed reactions (aside from the Michaelis complex) occur subsequently to the rate-determining transition state, and (ii) an acyl-enzyme intermediate, if such is formed, cannot be trapped with [(14)C]methanol in these systems.     

Identification of the site of acetyl-S-enzyme formation on avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase

Avian liver mitochondrial hydroxymethylglutaryl-CoA synthase contains an active-site cysteine involved in forming the labile acetyl-S-enzyme intermediate. Identification of and assignment of function to this cysteine have been accomplished by use of an experimental strategy that relies upon generation and rapid purification of the S-acetylcysteine-containing active-site peptide under mildly acidic conditions that stabilize the thioester adduct. Automated Edman degradation techniques indicate the peptide's sequence to be Arg-Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-Cys-Tyr. The acetylated cysteine corresponds to position 129 in the sequence deduced from cDNA data for the hamster cytosolic enzyme [Gil, G., Goldstein, J.L., Slaughter, C.A., & Brown, M.S. (1986) J. Biol. Chem. 261, 3710-3716]. The acetyl-peptide sequence overlaps that reported for a tryptic peptide that contains a cysteine targeted by the affinity label 3-chloropropionyl-CoA [Miziorko, H. M., & Behnke, C. E. (1985) J. Biol. Chem. 260, 13513-13516]. Thus, availability of these structural data allows unambiguous assignment of the acetylation site on the protein as well as a refinement of the mechanism explaining the previously observed affinity labeling of the enzyme.     

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E.T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants.(ABSTRACT TRUNCATED AT 250 WORDS)