Acyl silicates and acyl aluminates as activated intermediates in peptide formation on clays

Glycine reacts with heating on dried clays and other minerals to give peptides in much better yield than in the absence of mineral. This reaction was proposed to occur by way of an activated intermediate such as an acyl silicate or acyl aluminate (i.e., the anhydride of a carboxylic acid with Si–OH or Al–OH), analogous to acyl phosphates involved in several biochemical reactions including peptide bond synthesis. We confirmed the proposed mechanism by trapping the intermediate, as well as by direct spectroscopic observation of a related intermediate. The reaction of amino acids on periodically dried mineral surfaces represents a widespread, geologically realistic setting for prebiotic peptide formation viain situ activation.     

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.     

Acyl and amino intermediates in reactions catalyzed by thermolysin

Thermolysin showed peculiar transpeptidation reactions. Leu-Leu and/or Leu-Leu-Leu were produced at $\text{ca}$. pH 7 from Leu-Leu-NH₂ and Cbz-Leu-Leu. Isotope experiments indicated that the transpeptidation products did not use leucine released from the substrates as an acceptor. With Leu-Trp-Met, Leu-Leu, Leu-Leu-Leu and Met-Met were produced as transpeptidation products. A comparative study was done with α-chymotrypsin and pepsin. These results would indicate that thermolysin catalyzed reactions proceed via both acyl and amino intermediates depending upon the substrates, which has been proposed for the mechanism of pepsin. This may also be true in some cases for chymotrypsin and other proteases, which have been known as enzymes of the acyl-enzyme mechanism.     

Soluble fibrin-fibrinogen complexes as intermediates in fibrin gel formation

Oligomer formation in fibrinogen solutions following addition of thrombin was studied by addition of thrombin inhibitor at various times subsequent to thrombin, followed by size-exclusion chromatography (SEC) on a high-performance SEC column capable of resolving species of molecular weights less than or equal to 10(6). Peaks corresponding to species with 1, 2, 3, and 4 or more times the molecular weight of fibrinogen were detected and quantified via nonlinear least-squares curve-fitting procedures. The evolution of each of these peaks with time is well accounted for by a kinetic model in which the predominant component of each oligomeric molecular weight species is a linear complex of fibrinogen and fibrin. The observed predominance of trimeric over dimeric oligomers even at short times suggests that the thrombin-catalyzed release of the two A fibrinopeptides from a single molecule of fibrinogen is highly cooperative.     

Investigation of peptide thioester formation via N→Se acyl transfer

Native chemical ligation is widely used for the convergent synthesis of proteins. The peptide thioesters required for this process can be challenging to produce, particularly when using Fmoc‐based solid‐phase peptide synthesis. We have previously reported a route to peptide thioesters, following Fmoc solid‐phase peptide synthesis, via an N→S acyl shift that is initiated by the presence of a C‐terminal cysteine residue, under mildly acidic conditions. Under typical reaction conditions, we occasionally observed significant thioester hydrolysis as a consequence of long reaction times (~48 h) and sought to accelerate the reaction. Here, we present a faster route to peptide thioesters, by replacing the C‐terminal cysteine residue with selenocysteine and initiating thioester formation via an N→Se acyl shift. This modification allows thioester formation to take place at lower temperatures and on shorter time scales. We also demonstrate how application of this strategy also accelerates peptide cyclization, when a linear precursor is furnished with an N‐terminal cysteine and C‐terminal selenocysteine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Solid-phase acyl donor as a substrate pool in kinetically controlled protease-catalysed peptide synthesis

Recently we have demonstrated the advantage of solid- phase substrate pools mainly in equilibrium controlled protease-catalysed peptide syntheses. The extension of this approach to protease-catalysed acyl transfer reactions will be presented. The model reaction was systematically investigated according to both the influence of solid phases present in the system on enzyme activity as well as nucleophile concentration on peptide yield. The key parameter for obtaining high peptide yield via acyl transfer is the ratio between aminolysis and hydrolysis. We combined high nucleophile concentrations with solid-phase acyl donor pools. This approach enabled us to supply ester substrate and nucleophile in equimolar amounts in a high-density media without the addition of any organic solvent. Several multi-functional di- to tetrapeptides were obtained in moderate to high yields. ©1997 European Peptide Society and John Wiley & Sons, Ltd.     

Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate

Cholesterol esterification by acyl CoA:cholesterol acyl transferase (ACAT) in macrophages is a key process in atheroma foam cell formation. However, the process of cholesterol substrate delivery to ACAT is not well defined. In this study, J774 macrophages, which form foam cells with native low density lipoprotein (LDL), were labeled with [3H]cholesterol-containing liposomes. Most (80-90%) of the cholesterol label could be converted by cholesterol oxidase to cholestenone, suggesting plasma membrane localization; only 0.6% of the label was in cholesteryl ester (CE). In cells chased for 6 h in medium lacking LDL, the distribution of label was essentially unchanged, whereas in cells chased with LDL, 28% of the label was incorporated into CE concomitant with a decrease in cholestenone label to 50%. [3H]Cholesterol-labeled mouse peritoneal macrophages incubated with acetyl-LDL, and both J774 and mouse peritoneal macrophages incubated with 25-hydroxy-cholesterol, also showed a shift of label from cholestenone to CE. Similar results were found when cellular cholesterol was biosynthetically labeled with [3H]mevalonate. The percentage of cholesterol substrate for ACAT in LDL-treated J774 macrophages which originates from endogenous cellular pools (versus that originating from LDL itself) is approximately 50%. We conclude that upon activation of ACAT in macrophages, there is a novel process whereby a cholesterol oxidase-accessible pool of cellular cholesterol, presumably plasma membrane cholesterol, is translocated to ACAT in the endoplasmic reticulum.     

The possible role of clays in prebiotic peptide synthesis

Hypotheses of macromolecule formations during the prebiotic era are described. The presumed role of minerals and clays in these reactions are: concentration of monomers, proton release by ion exchange whenever the reaction demands it, scattering of the charges of the interacting substances, thus allowing such substances to interact, which in the absence of clays repel each other due to their charges. Because of these reasons the polymerization mechanism in the presence of clays is different from that in their absence. While in the absence of clays only free amino acids or peptides can interact with active amino acid anhydrides, giving thus peptides increased by only one unit, in the presence of clays two molecules of amino acid anhydrides can interact, giving a still active peptide anhydride which can interact with another active peptide. Clays catalyze polymerization only in these cases where the amino acid is small enough to enter between the sheets of the clay. Apparently most of the reactions also occur there and not on the surface of the clay. Copolymerization of different pairs of amino acids proceeds selectively in the presence of clay. The relationship between this selecitvity and prebiotic parent proteins is discussed.     

Acyl CoA:Cholesterol acyl transferase activity in human placental microsomes: Inhibition by progesterone

The characteristics of acyl CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) in microsomes prepared from human term placenta were studied and the rate of incorporation of [1-¹⁴C] oleoyl CoA into cholesteryl esters was measured. The apparent K_m of the enzyme for [1-¹⁴C] oleoyl CoA was 38 ± 9 μm and the V for the reaction was 15 ± 6 pmol × mg^? protein × min^?1. The Hill coefficient for the reaction was 1.2, indicative of some degree of positive cooperativity. Cholesterol, added to the incubation mixture, did not influence ACAT activity, indicating that endogenous microsomal cholesterol served as an effective substrate for the placental ACAT enzyme. However, [1,2-³H]cholesterol in the presence of oleoyl CoA was incorporated into cholesteryl esters by placental microsomes. When progesterone was present in the incubation mixture at a concentration of 20 μm, ACAT activity was inhibited 50%. Pregnenolone, 5α-dihydroprogesterone, 17α-hydroxyprogesterone, deoxycorticosterone, dehydroisoandrosterone, androstenedione, testosterone, and estradiol-17β also inhibited ACAT activity, whereas corticosterone, cortisol, and estriol had little effect. These results are supportive of the view that ACAT activity in human placenta may be regulated by endogenously synthesized steroid hormones.     

Acidic metabolites. VI. 20 alpha-isosteroids as intermediates in 20 alpha-dihydrosteroid formation

We have previously shown that human subjects metabolize the 20 beta-epimer of isocortisol (11 beta, 17,20 beta-trihydroxy-3-oxo-pregn-4-en-21-al) to both 20 alpha- and 20 beta-hydroxy steroid end products. In this paper we describe the synthesis of tritium labeled 20 alpha-epimers of isocortisol and isoTHF (3 alpha, 11 beta, 17,20 alpha-tetrahydroxy-5 beta-pregnan-21-al) and their metabolic fate in humans. Both steroids yielded 20 alpha-hydroxy urinary neutral end-products (cortols and cortolones) and no 20 beta-hydroxy epimers. Regeneration of 17-ketols from aldols occurred to a small extent with isoTHF, but not with isocortisol. Isocortisol and isoTHF yielded less cortoic acids than did the corresponding ketols. The results provide further evidence that in man the stereochemistry at C-20 of the end-products of corticosteroid metabolism is determined by the configuration of the aldol at C-20 prior to subsequent metabolic events.     

Trypsin-catalyzed peptide synthesis withm-guanidinophenyl andm-(guanidinomethyl)phenyl esters as acyl donor component

Summary Two series of inverse substrates,m-guanidinophenyl andm-(guanidinomethyl)phenyl esters derived fromN-(tert-butyloxycarbonyl)amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis. The kinetic behavior of these esters toward tryptic hydrolysis was analyzed. They were found to couple with an acyl acceptor such asl-alaninep-nitroanilide to produce dipeptide in the presence of trypsin.Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. Within the enzymatic peptide coupling methods, this approach was shown to be advantageous, since the resulting peptides are resistant to the enzymatic hydrolysis.Abbreviations Boc tert-butyloxycarbonyl - Aib -aminoisobutyric acid - DMSO dimethylsulfoxide - Tris tris(hydroxymethyl)aminomethane - MOPS 3-morpholino-l-prop anesulfonate - G guanidinophenyl - GM (guanidinomethyl)phenyl - pNA p-nitroanilide     

Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products.

The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.     

The formation of lipid-linked sugars as intermediates in glycoprotein synthesis in rabbit mammary gland

1. The incorporation of d-[1-(14)C]mannose, d-[2-(3)H]mannose and N-acetyl-d-[1-(14)C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-(3)H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-(14)C]mannose and N-acetyl-d-[1-(14)C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-(14)C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000-80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-(14)C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-(14)C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.     

Acyl intermediates in penicillopepsin-catalysed reactions and a discussion of the mechanism of action of pepsins.

Penicillopepsin catalyses transpeptidation reactions involving the transfer of the N-terminal amino acids of suitable substrates via covalent acyl intermediates to acceptor peptides, usually the substrate. The major products obtained when Phe-Tyr-Thr-Pro-Lys-Ala and Met-Leu-Gly were used as substrates were Phe-Phe and Met-Met respectively. With Met-Leu-Gly the tetrapeptide Met-Met-Leu-Gly was observed as probable intermediate. Co-incubation of Leu-Tyr-Leu and Phe-Tyr-Thr-Pro-Lys-Ala led to the formation of Leu-Phe and Phe-Leu as well as Leu-Leu and Phe-Phe. No reaction was observed with tripeptides in which the first or second amino acid is glycine. It appears that two amino aicds with large hydrophobic residues are needed for the transpeptidation reaction. Nucleophilic compounds other than peptides, such as hydroxylamine, aliphatic alcohols and dinitrophenylhydrazine, were not acceptors for the acyl group. Leucine, phenylalanine and leucine methyl ester also had no effect on the reaction. The transpeptidation reaction proceeded readily at pH 3.6 and 4.7. At pH 6.0 the reaction was slow and at pH 1.9 little or no transpeptidation was observed. Porcine pepsin catalyses similar transpeptidation reactions. Sequence studies show that porcine pepsin and penicillopepsin are homologous. The present study also suggests that they have a very similar mechanism. Evidence available at this time indicates that the mechanism of these enzymes is complex and may be modulated by secondary substrate-enzyme interactions. A hypothesis is presented which proposes that pepsin-catalysed reactions proceed via different covalent intermediates (amino-intermediates or acylintermediates) depending on the nature of the substrate. The possibility that some reactions do not involve covalent intermediates is also discussed.