Studies of oligoadenylate formation on a poly(U) template

We have studied a variety of condensation reactions involving poly (U) as template and isomeric adenosine dinucleotides as substrates. We find that [3'-5']-linked dinucleotides such as A3pA and pA3pA are better acceptors than the corresponding [2'-5']-linked compounds, while ImpA2pA is a better donor than ImpA3pA. The reaction between A2pA and ImpA3pA, for example, yields only 4% of product while the reaction of A3pA with ImpA2pA yields 86% of product. The more efficient condensation reactions of dimers are about as efficient as the self-condensation of ImpA. They yield a few percent of material in which five or more substrate molecules are linked together. The percentage of the natural [3'-5']-linkage in the product varies greatly, from as little as 1% to as much as 45%.     

The partial molar heat capacity and volume of the peptide backbone group of proteins in aqueous solution

The partial molar heat capacities have been determined for the series of peptides alanyl(glycyl)(x)glycine, x=1-3, and for the compounds N-acetylglycinamide and N-acetyl glycylglycinamide in aqueous solution over the temperature range 10-100 degrees C using high sensitivity scanning microcalorimetry. The partial molar volumes for these compounds have also been determined over the temperature range from 10 to 90 degrees C using a scanning densimetric method. The results were used to derive the partial molar heat capacities and volumes of the glycyl group at temperatures in the range 10-100 degrees C. The results obtained are critically compared with literature results derived using heat capacity and volume data for some oligoglycines.     

Prebiotic peptide-formation in the solid state

The reactions of glycine with inorganic polyphosphates in the solid state have been studied. The formation of peptides up to the decamer occurs at moderate temperatures(r.t.-100 degrees C) in the presence of imidazole and magnesium chloride. If adenosine 5' -monophosphate is added to the reaction mixture, 2'(3') -o-glycyl adenosine 5'-monophosphate is also obtained. These reactions could have occurred on the primitive earth.     

Catalysis of peptide bond formation by histidyl-histidine in a fluctuating clay environment

Summary The condensation of glycine to form oligoglycines during wet-dry fluctuations on clay surfaces was enhanced up to threefold or greater by small amounts of histidyl-histidine. In addition, higher relative yields of the longer oligomers were produced. Other specific dipeptides tested gave no enhancement, and imidazole, histidine, and N-acetylhistidine gave only slight enhancements. Histidyl-histidine apparently acts as a true catalyst (in the sense of repeatedly catalyzing the reaction), since up to 52 nmol of additional glycine were incorporated into oligoglycine for each nmol of catalyst added. This is the first known instance of a peptide or similar molecule demonstrating a catalytic turnover number greater than unity in a prebiotic oligomer synthesis reaction, and suggests that histidyl-histidine is a model for a primitive prebiotic protoenzyme. Catalysis of peptide bond synthesis by a molecule which is itself a peptide implies that related systems may be capable of exhibiting autocatalytic growth.     

Montmorillonite,Oligonucleotides, RNA and Origin of Life

Na-montmorillonite prepared from Volclay by the titration method facilitates the self-condensation of ImpA, the 5'-phosphorimidazolide derivative of adenosine. As was shown by AE-HPLC analysis and selective enzymatic hydrolysis of products, oligo(A)s formed in this reaction are 10 monomer units long and contain 67% 3',5'-phosphodiester bonds (Ferris and Ertem, 1992a). Under the same reaction conditions, 5'-phosphorimidazolide derivatives of cytidine, uridine and guanosine also undergo self-condensation producing oligomers containing up to 12-14 monomer units for oligo(C)s to 6 monomer units for oligo(G)s. In oligo(C)s and oligo(U)s, 75-80% of the monomers are linked by 2',5'-phosphodiester bonds. Hexamer and higher oligomers isolated from synthetic oligo(C)s formed by montmorillonite catalysis, which contain both 3',5'- and 2',5'-linkages, serve as catalysts for the non-enzymatic template directed synthesis of oligo(G)s from activated monomer 2-MeImpG, guanosine 5'-phospho-2-methylimidazolide (Ertem and Ferris, 1996). Pentamer and higher oligomers containing exclusively 2',5'-linkages, which were isolated from the synthetic oligo(C)s, also serve as templates and produce oligo(G)s with both 2',5'- and 3',5'-phosphodiester bonds. Kinetic studies on montmorillonite catalyzed elongation rates of oligomers using the computer program SIMFIT demonstrated that the rate constants for the formation of oligo(A)s increased in the order of 2-mer < 3-mer < 4-mer ... < 7-mer (Kawamura and Ferris, 1994). A decameric primer, dA(pdA)8pA bound to montmorillonite was elongated to contain up to 50 monomer units by daily addition of activated monomer ImpA to the reaction mixture (Ferris, Hill and Orgel, 1996). Analysis of dimer fractions formed in the montmorillonite catalyzed reaction of binary and quaternary mixtures of ImpA, ImpC, 2-MeImpG and ImpU suggested that only a limited number of oligomers could have formed on the primitive Earth rather than equal amounts of all possible isomers (Ertem and Ferris, 2000). Formation of phosphodiester bonds between mononucleotides by montmorillonite catalysis is a fascinating discovery, and a significant step forward in efforts to find out how the first RNA-like oligomers might have formed in the course of chemical evolution. However, as has been pointed out in several publications, these systems should be regarded as models rather than a literal representation of prebiotic chemistry (Orgel, 1998; Joyce and Orgel, 1999; Schwartz, 1999).     

Use of potato tuber nucleotide pyrophosphatase to synthesize adenosine 5'-monophosphate methyl ester: evidence that the solvolytic preferences of the enzyme are regulated by pH and temperature

Nucleotide alkyl esters are pharmacologically important as potential (ant)agonists of purinoceptors and inhibitors of enzymes. Potato nucleotide pyrophosphatase (PNP) was compared with snake venom phosphodiesterase (SVP) as a catalyst to synthesize nucleotide alkyl esters. In methanol-water mixtures, the methanolysis/hydrolysis ratio of PNP, but not SVP, changed with pH and temperature, being optimal at high pH and low temperature. In a semi-preparative experiment, a crude PNP preparation produced 0.17 mM AMP-O-methyl ester (AMP-OMe) from 1 mM diadenosine 5',5"'-P1,P2-diphosphate (AppA) and 5M methanol, at pH 9 and 0 degrees C. Drawbacks to large-scale use are: low rates inherent to low temperatures, ATP unsuitability as a substrate for alcoholysis, and high cost of AppA. Advantages of PNP vs. SVP are cheapness, non-toxicity, and availability of the enzyme source.     

Diketopiperazine-mediated peptide formation in aqueous solution

Though diketopiperazines (DKP) are formed in most experiments concerning the prebiotic peptide formation, the molecules have not been paid attention in the studies of chemical evolution. We have found that triglycine, tetraglycine or pentaglycine are formed in aqueous solution of glycine anhydride (DKP) and glycine, diglycine or triglycine, respectively. A reaction of alanine with DKP resulted in the formation of glycylglycylalanine under the same conditions. These results indicate that the formation of the peptide bonds proceeds through the nucleophilic attack of an amino group of the amino acids or the oligoglycines on the DKP accompanied by the ring-opening.The formation of glycine anhydride, di-, tri- and tetraglycine was also observed in a mixed aqueous solution of urea and glycine in an open system to allow the evaporation of ammonia. A probable pathway is proposed for prebiotic peptide formation through diketopiperazine on the primitive Earth.     

Diketopiperazine-mediated peptide formation in aqueous solution II. Catalytic effect of phosphate

The previous paper (I) reported that DKP (glycine anhydride) spontaneously reacts with glycine (Gly) or oligoglycines (Gly _n ) to produce longer oligoglycines (Gly _n+2). This paper presents that phosphate catalyzes the condensation reaction quite effectively.Formation of Gly₄ from DKP (0.1 M) and Gly₂ (0.1 M) in phosphate solution of various concentrations was investigated at a neutral pH at 41 °C. The yields of Gly₄ increased almost linearly with the concentration of phosphate from 0.06 M to 0.24 M. The yield in 0.24 M phosphate solution was approximately one hundred times as high as that in the absence of the phosphate, whereas in the case of Gly₃ formation from DKP and Gly the effect of the phosphate was of ten times lower than in the former case. Orthophosphate was the most effective catalyst among the various kind of chemicals tried in the present investigation including polyphosphates.     

Cyanamide mediated syntheses under plausible primitive earth conditions

The synthesis of palmitoylglycerols in good yields occurs when a solution of glycerol, ammonium palmitate, cyanamide and imidazole is dried and heated at ambient humidity at temperatures ranging from 60 degrees--100 degrees C for 16 h. Much less product is formed in the absence of either or both cyanamide or imidazole. This work suggests that acylglycerols could have been synthesized on the primitive Earth under plausible prebiotic conditions which were similar but not identical to those which have been shown to condense deoxynucleotides into oligodeoxynucleotides and amino acids into peptides.     

Inactivation of human fibroblast collagenase by chloroacetyl N-hydroxypeptide derivatives.

When human fibroblast collagenase was incubated with ClCH2CO-(N-OH)Leu-Ala-Gly-NH2 (2-5 mM) in Tris buffer, pH 7.4 at 25 degrees C, a slow, time-dependent inhibition of the enzyme was observed. Dialysis against a buffer to remove free inhibitor did not reactivate the enzyme. A reversible competitive inhibitor, phthaloyl-GlyP-Ile-Trp-NHBzl (50 microM) partially protected the enzyme from inactivation by the compound. From the concentration dependent rates of inactivation Ki = 0.5 +/- 0.1 mM and k3, the rate constant for inactivation = 3.4 +/- 0.3 x 10(-3) min-1 were determined. The inactivation followed the pH optimum (6.5-7.0) for the enzyme activity, suggesting direct involvement of the same active site residue(s). The reaction mode of the inhibitor may be analogous to that of the inactivation of Pseudomonas aeruginosa elastase [Nishino, N. and Powers, J. (1980) J. Biol. Chem., 255, 3482] in which the catalytic glutamate carboxyl was alkylated by the inhibitor after its binding to enzyme through the hydroxamic Zn2+ ligand. All carboxyl groups in the inactivated collagenase were modified with 0.1 M ethyl dimethylaminopropyl carbodiimide/0.5 M glycinamide in 4 M guanidine at pH 5. The inactivator-affected carboxyl group was then regenerated with 1 M imidazole at pH 8.9, 37 degrees C for 12 h and the protein was radiolabeled with 3H-glycine methyl ester and carbodiimide to incorporate 0.9 residue glycine per mol enzyme.     

Polymerization of amino acid methyl esters via their copper complexes.

Polymerization of glycine methyl ester catalyzed by cupric ions in organic solvents yields oligoglycines with a degree of polymerization up to none. With a trifunctional amino acid, the yield and degree of polymerization were much lower. Extension of this reaction to an aqueous medium was not successful even when copper ions were complexed with substances like montmorillonite or fatty acids. The prebiotic significance of this reaction is discussed.     

Mechanism of pigeon liver malic enzyme: modification of essential carboxyl groups

The maximum velocity of the reaction catalyzed by the pigeon liver malic enzyme depends on the ionization of a functional group of pKa 6.7. This pKa value is independent of temperature within the range 30 degrees-49 degrees C, suggesting the ionization of a carboxyl group. The enzyme activity is inactivated by N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K) at pH 6.0 and 25 degrees C. N-Methylhydroxamine regenerates the enzymatic activity whereas glycine ethyl ester does not. The addition of Mn2+, NADP+, and L-malate to the incubation mixture decreases the inactivation rate, suggesting that the reaction takes place in the active center. The binding capacities of the modified enzyme with NADP+, L-malate, pyruvate, and Mn2+ are not impaired. The kinetic and chemical evidence indicates that the inactivation is due to the modification of a carboxyl group which may be from glutamyl or aspartyl residues of the enzyme. This carboxyl group might function as a general acid-base catalyst. A detailed mechanism in terms of the exact amino acid residues involved is proposed.     

Spontaneous and promoted association of linear oligoglycines

Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was observed for free oligoglycines H-(Gly)m-OH(m = 3-5). At the same time, oligoglycylamides H-(Gly)m-NH2 (m = 3-5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an additional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolecular clustering of peptide chains, as expected, was observed. This means that the presence of several oligoglycine chains bound to each other in one center is not a necessary prerequisite for polyglycine II-type association.     

Condensation of oligoglycines with trimeta- and tetrametaphosphate in aqueous solutions

The dehydration condensation of glycine with trimetaphosphate in aqueous solution has been reinvestigated. Although it has been reported that the condensation of glycine under the alkaline conditions was brought about through the formation of cyclic acylphosphoramidate and hence the condensation of polyglycines could not occur, we found that the condensation of oligoglycines with trimeta- and tetrametaphosphate in aqueous solution are possible through the formation of their acylphosphates under the neutral or weak acidic conditions.Aqueous solutions of 1.0 M glycylglycine and 1.0 M trimetaphosphate in the various pH from 4.0 to 9.0 were incubated at 38 °C. The solutions were analyzed by HPLC with ninhydrin reaction system. Tetraglycine and hexaglycine were detected and their maximum yields were given in the reaction carried out around pH 7. They are approximately 15% and 4% after 30 days, respectively. Analogous experiments were performed with tetrametaphosphate. The results showed a similar pH dependence for the condensation, but the yields were about one-tenth of those of corresponding experiments with trimetaphosphate.Relative rates of dimerization of glycine, diglycine and triglycine in the equimolar concentration were also investigated at pH 6.0 at 38 °C. The rates for digylcine and triglycine were approximately twice and four times as large as that for glycine.Relevance of the experiments to chemical evolution is discussed.     

Polymerization of amino acid methyl esters via their copper complexes

Polymerization of glycine methyl ester catalyzed by cupric ions in organic solvents yields oligoglycines with a degree of polymerization up to nine. With a trifunctional amino acid, the yeild and degree of polymerization were much lower. Extension of this reaction to an aqueous medium was not successful even when copper ions were complexed with substances like montmorillonite or fatty acids. The prebiotic significance of this reaction is discussed.Contribution to the 4th International Conference on the Origin of Life, Barcelona, June 1973.     

Interactions of Neurospora crassa plasma membrane H+-ATPase with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline

The carboxyl group activating reagent N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) interacts with the Neurospora plasma membrane H+-ATPase in at least three different ways. This reagent irreversibly inhibits ATP hydrolysis with kinetics that are pseudo-first-order at several concentrations of EEDQ, and an appropriate transform of these data suggests that 1 mol of EEDQ inactivates 1 mol of the H+-ATPase. Inhibition probably involves activation of an ATPase carboxyl group followed by a nucleophilic attack by a vicinal nucleophilic functional group in the ATPase polypeptide chain, resulting in an intramolecular cross-link. The enzyme is protected against EEDQ inhibition by MgATP in the presence of vanadate, a combination of ligands that has previously been shown to "lock" the H+-ATPase in a conformation that presumably resembles the transition states of the enzyme phosphorylation and dephosphorylation reactions, but is not protected by the substrate analogue MgADP, which is consistent with the notion that one or both of the residues involved in the EEDQ-dependent inhibitory intramolecular cross-linking reaction normally participate in the transfer of the gamma-phosphoryl group of ATP, or are near those that do. The ATPase is also labeled by the exogenous nucleophile [14C]glycine ethyl ester in an EEDQ-dependent reaction, and the labeling is diminished in the presence of MgATP plus vanadate. However, peptide maps of [14C]glycine ethyl ester labeled ATPase demonstrate that the labeling is not related to the EEDQ inhibition reaction in any simple way.(ABSTRACT TRUNCATED AT 250 WORDS)     

Michael addition of imidazole with acrylates catalyzed by alkaline protease from Bacillus subtilis in organic media

A new activity of alkaline protease from Bacillus subtilis for Michael addition reactions of imidazole, 4-nitro-1H-imidazole and 2-methyl-4-nitro-1H-imidazole with acrylates and acrylic acid was investigated. The reactions were carried out in pyridine at 50 degrees C for 72 h. Five N-substituted imidazole derivatives were obtained using acrylate esters, but not acrylic acid, in yields from 62% to 76%.     

Chiral Selection in Oligoadenylate Formation in the Presence of a Metal ion Catalyst or Poly(U) Template

The lead ion-catalyzed oligomerization of 5′-phosphorimidazolides of D-, L- or racemic DL-adenosine (D-ImpA, L-ImpA and DL-ImpA) gave oligoadenylates up to a pentamer. The oligomers resulting from racemic ImpA were comparable in yields and length to those from chiral D- or L-ImpA. A complex mixture of homochiral and heterochiral oligomers was formed in the reaction from racemic ImpA. Total dimer product from racemic ImpA by the lead ion catalyst showed homochiral selectivity. The reaction catalyzed by uranyl ion yielded oligoadenylates up to 15mer from chiral D- or L-ImpA in over 95% yield. A complex mixture of isomeric oligoadenylates was formed from racemic DL-ImpA in the presence of uranyl ion catalyst in comparable yields to those from D- or L-ImpA. The analysis of the dimer product from DL-ImpA showed that the homochiral 2′ –5′ linked dimer was selectively formed. D-ImpA polymerized effectively on a poly(U) template, which is exclusively composed of D-uridine, yielding oligoadenylates up to a pentamer. In contrast, L-ImpA or racemic DL-ImpA polymerized far less efficiently on the poly(U) template, demonstrating that chiral selection takes place in the poly(U) template-directed oligoadenylate formation.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli

Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.