Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase,a multifunctional polyketide biosynthetic enzyme,by free coenzyme A

6-Hydroxymellein (6HM) synthase is a multifunctional polyketide enzyme induced in carrot cells, whose fully active homodimer catalyzes condensation of acyl-CoAs and the NADPH-dependent ketoreduction of the enzyme-bound intermediate. 6HM-forming activity of the synthase was markedly decreased when the reaction mixture pH was adjusted from 7.5 to 6.0. However, under these slightly acidic conditions, the acyl condensation catalyzed by the dissociated monomer enzyme was appreciably stimulated by addition of free coenzyme A (CoA). In contrast, the condensation reaction at pH 6.0 was significantly inhibited in the presence of CoA when the reaction was carried out with the NADPH-omitted dimer synthase. Among the kinetic parameters of the acyl condensation, velocity of the monomer-catalyzing reaction at the acidic pH was appreciably increased upon addition of CoA while K(m)s did not show any significant change in the presence and absence of the compound. These results suggest that CoA associates with a specific site in the dissociated monomeric form of 6HM synthase, and the velocity of the acyl condensation reaction catalyzed by the CoA-synthase complex appreciably increases in acidic conditions.     

Proposed mechanism for the condensation reaction of citrate synthase: 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A

The crystal structure of the ternary complex citrate synthase-oxaloacetate-carboxymethyl coenzyme A has been solved to a resolution of 1.9 A and refined to a conventional crystallographic R factor of 0.185. The structure resembles a proposed transition state of the condensation reaction and suggests that the condensation reaction proceeds through a neutral enol rather than an enolate intermediate. A mechanism for the condensation reaction is proposed which involves the participation of three key catalytic groups (Asp 375, His 274, and His 320) in two distinct steps. The proposed mechanism invokes concerted general acid-base catalysis twice to explain both the energetics of the reaction and the experimentally observed inversion of stereochemistry at the attacking carbon atom.     

Dynamics of DNA condensation

The condensation of DNA induced by spermine and spermidine is investigated by equilibrium titrations and stopped-flow and field-jump experiments using scattered light detection. The spermine concentration required for the cooperative condensation process is measured at different DNA concentrations; these data are used to evaluate both the condensation threshold degree of spermine binding and the binding constant of spermine according to an excluded-site model. Stopped-flow measurements of the spermine-induced condensation demonstrate the existence of two processes: (1) A "fast" reaction is observed in the millisecond time range, when the reactant concentrations are around 1 microM; it is associated with a characteristic induction period and is assigned to the intramolecular condensation reaction. (2) A slow reaction with time constants of, e.g., 100 s strongly dependent upon both spermine and DNA concentrations is assigned to an intermolecular DNA association. The unusual time course of the intramolecular condensation reaction with the induction period provides evidence for a "threshold kinetics". During the induction period, spermine molecules are bound to DNA, but the degree of binding remains below the threshold value. As soon as the degree of ligand binding arrives at the threshold, the DNA is condensed in a relatively fast reaction. Model calculations of the spermine binding kinetics according to an excluded-site model demonstrate that the spermine molecules bound to DNA are mobile along the double helix. A comparison of the experimental data with the results of Monte Carlo simulations suggests a rate constant of approximately 200 s-1 for spermine movement by one nucleotide residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of a fatty acid elongase ELO-like condensing enzyme

The condensation step of fatty acid elongation is the addition of a C₂ unit from malonyl-CoA to an acyl primer catalyzed by one of two families of enzymes, the 3-ketoacyl-CoA synthases and the ELO-like condensing enzymes. 3-Ketoacyl-CoA synthases use a Claisen-like reaction mechanism while the mechanism of the ELO-catalyzed condensation reaction is unknown. We have used site-directed mutagenesis of Dictyostelium discoideum EloA to identify residues important to catalytic activity and/or structure. Mutation of highly conserved polar residues to alanine resulted in an inactive enzyme strongly suggesting that these residues play a role in the condensation reaction.     

Condensation of amino acids to form peptides in aqueous solution induced by the oxidation of sulfur(iv): An oxidative model for prebiotic peptide formation

Condensation of amino acids to peptides is an important step during the origin of life. However, up to now, successful explanations for plausible prebiotic peptide formation pathways have been limited. Here we report that the oxidation of sulfur (IV) can induce the condensation reaction of carboxylic acids and amines to form amides, and the condensation reaction of amino acids to form peptides. This might be a general reaction contributing to prebiotic peptide formation.     

Condensation reaction between angiotensin II and dicarboxylic acid in water at high temperature without any catalytic agent additive

The condensation reaction of angiotensin II and tartaric acid in water at 100-140 degrees C proceeded without any added catalytic agent. One of the products, N-CO-tartarylangiotensin II, was confirmed by LC-MS, positive-ion MALDI-MS, and the fluorescamine method as a condensation product. The initial concentration of the substrates, temperatures, and initial pH affected the yield of the product. The higher concentrations and temperatures increased the concentration of the condensation product. The optimal pH for the condensation reaction was 3.0. The other C4 saturated dicarboxylic acids, malic and succinic acids, were also condensed by angiotensin II.     

Thermodynamics of peptide bond formation at clay mineral surfaces

Summary The possibility of surface catalysed condensation of unsubstituted amino acids on kaolinite in aqueous systems at elevated temperatures was investigated; no evidence of clay catalysed polycondensation has been found. The thermodynamic feasibility of the hypothetical lysine/dilysine condensation reaction in the temperature-range up to 90° C was evaluated for a range of experimental conditions by the combination of measured free energies of lysine/dilysine cation exchange on kaolinite and on montmorillonite, and free energies for the analogous condensation reaction in homogeneous solution. The results indicate that, in spite of the high selectivity of the clays for the adsorption of cation dimers from dilute solutions, the thermodynamic barrier to the surface condensation of unsubstituted amino acids on clay minerals in aqueous systems up to 90° C is not lower than it is in homogeneous solution.     

鸭肝脂肪酸合成酶的NADPH底物抑制及作用动力学

己知动物脂肪酸合成酶的底物乙酰辅酶Ａ和丙二酰辅酶Ａ具有竞争性双底物抑制的乒乓机制。实验发现鸭肝脂肪酸合成酶的第三个底物ＮＡＤＰＨ也具有底物抑制,并研究了它的规律及与ＮＡＤＰＨ有关的稳态动力学。发现对于该酶的全反应,增加丙二酰辅酶Ａ浓度,降低环境盐浓度,均使ＮＡＤＰＨ底物抑制减少。但以ＮＡＤＰＨ作底物的酮酰还原和烯酰还原二步单独反应以及包含四步单独反应的乙酰乙酰辅酶Ａ还原反应都无ＮＡＤＰＨ底物抑制现象。ＮＡＤＰＨ底物抑制对丙二酰辅酶Ａ为竞争性,丙二酰辅酶Ａ底物抑制对ＮＡＤＰＨ为非竞争性。在全反应中ＮＡＤＰＨ和丙二酰辅酶Ａ之间发现为乒乓机制,在乙酰乙酰辅酶Ａ还原反应中,两个底物ＮＡＤＰＨ和乙酰乙酰辅酶Ａ之间则表现为序列反应机制。降低环境盐浓度使ＮＡＤＰＨ和丙二酰辅酶Ａ之间的乒乓机制向序列机制转化。在全反应中,ＮＡＤＰ产物抑制相对ＮＡＤＰ为竞争性,对丙二酰辅酶Ａ为非竞争性。     

The effect of hemoglobin ligands on the kinetics of human hemoglobin A1c formation

HbA1c is the most prevalent of the minor human hemoglobins. It is formed by the nonenzymatic addition of glucose to the alpha-amino group of the beta chain by an initial condensation reaction and a subsequent intermolecular Amadori rearrangement. We have developed a method of analysis which utilizes high performance liquid chromatography to follow the formation of HbA1c and greatly simplifies the determination of the kinetic parameters associated with this reaction. This has allowed us to study the effects of several Hb ligands, including the hydrogen ion, on the kinetics of this glycosylation reaction. Both the initial condensation reaction and the subsequent rearrangement are shown to exhibit acid catalysis, but the rate of the condensation step is limited by the extent of protonation of the alpha-amino group. The variation in kinetic parameters as a function of hydrogen ion concentration has allowed us to determine the probable reaction mechanism of HbA1c formation by comparison to previously reported model systems of Schiff base formation and Amadori rearrangement. The formation of pre-HbA1c from deoxy-Hb shows an increased forward rate when compared to oxy-Hb. The presence of physiologic concentrations of CO2 causes a proportional decrease in both k1 and k-1. 2,3-Diphosphoglycerate causes a significant increase in the keq of the formation reaction. The effects of CO and the substitution of L-glucose for D-glucose are not significant.     

Penicillin acylase catalyzed synthesis of penicillin-G from substrates anchored in cyclodextrins

Penicillin acylase (EC 3.5.1.11) catalyses the condensation of phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA) to form benzylpenicillin (BP). Both PAA and 6-APA were found to form host-guest complexes with beta-methylcyclodextrin (beta m-CD) and gamma-cyclodextrin (gamma-CD) respectively. The rate of the reaction catalyzed by the enzyme remained unaffected if one of the substrates used was in the cyclodextrin complexed form. However, in this case, the reaction lasted longer and yielded about 20 per cent more products compared to the condensation reaction involving only uncomplexed substrates. There was distinct increase in the rate of formation of the antibiotic, if both substrates used are in CD-complexed form.     

In vitro condensation of amino acids in aqueous media: A synthesis driven by catalytic carbon monoxide oxidation

A novel mechanism is proposed for amino acid condensations to oligopeptides in aqueous media. The palladium-catalyzed condensation appears to proceed through a mechanism involving the Pd(0)/Pd(II) redox cycle. The reaction requires the use of carbon monoxide and an oxidant, and it is proposed that the oxidation of carbon monoxide to carbon dioxide drives the otherwise thermodynamically uphill condensation. The reaction occurs for several amino acids including glycine, alanine, β-alanine, and 5-aminopentanoic acid. Competition studies between glycine and alanine reveal steric effects on product formation. Dipeptides are the predominant condensation products, though some longer chains, containing up to 4 amino acids, can be observed. First-order plots for dipeptide appearance show an inverse, secondary, isotope effect consistent with the rate determining carboxylic-carbamic anhydride formation.     

l-Methyl-Z-(2′-Hydroxyphenyl)Imidazole–A Catalytic Phosphate protecting group in deoxyoligonucleotide synthesis

Abstract

The rate of condensation using the phosphate triester method of deoxyoligonucleotide synthesis is dramatically increased by the introduction of a phosphate protecting group bearing a nuclcophilic catalyst in the proper position. Following condensation (resulting in the formation of a phosphate triester) the catalytic protecting group can be removed leaving a dinucleotide, or the condensation reaction can be repeated to synthesize an oligonucleotide. This development is a significant advance in the chemical synthesis of deoxyoligonucleotides.     

alpha-Chymotrypsin in plastein synthesis: influence of substrate concentration on enzyme activity.

The role of alpha-chymotrypsin in the plastein reaction was studied using a peptic hydrolysate of albumin as substrate. Study of this reaction simultaneously by different methods showed that the plastein reaction is enzyme catalyzed and is highly dependent on environmental conditions. A gel permeation chromatography study of the plastein reaction showed simultaneous increases in the high- and low-molecular-weight oligopeptide fractions; a transpeptidation mechanism may be involved in the reaction. A study of the effect of substrate concentration on the plastein reaction catalyzed by alpha-chymotrypsin showed a profile with both hydrolytic and synthetic activities. This effect was also observed when the reaction course was followed by quantification of the free amino groups at different substrate concentrations, showing that a condensation mechanism is responsible for the synthetic activity when the substrate concentration is very high. These results have led us to conclude that the plastein reaction involves a transpeptidation and/or condensation mechanism, which is a function of the substrate concentration.     

β-Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism

1. beta-Ketothiolase was purified 49-fold from fructose-grown cells of Hydrogenomonas eutropha H16 with a yield of 27%; the purification procedure involved precipitation by cetyltrimethylammonium bromide, DEAE-cellulose chromatography and exclusion chromatography on Sephadex G-200; the freeze-dried enzyme is stable. The molecular weight determined by sucrose-gradient centrifugation (8.2S) and by gel filtration is 147000-150000. The optimum pH for the cleavage reaction is 8.1, that for the condensation reaction 7.8, both measured in Tris-HCl buffer. 2. The kinetics of the cleavage reaction are described. Substrate-saturation curves were measured with both acetoacetyl-CoA and CoA as the variable substrates. The concentration of the second substrate was kept constant and was varied during successive experiments. The cleavage reaction is characterized by substrate inhibition by acetoacetyl-CoA, which is partially relieved by free CoA. Hill plots indicate two acetoacetyl-CoA-binding sites. 3. The substrate(acetyl-CoA)-saturation curve for the condensation reaction is hyperbolic. The K(m) was 3.9x10(-4)m-acetyl-CoA. In the presence of CoA sigmoidal curves were obtained, with an increasing sigmoidicity from 0.03 to 0.30mm-CoA. The inhibitory action of CoA on the beta-ketothiolase condensation reaction and its possible involvement in the regulation of poly-beta-hydroxybutyrate synthesis and degradation are discussed.     

Specificity of pepsin-catalyzed peptide bond synthesis

The rates of the pepsin-catalyzed synthesis of oligopeptides of the general type A-Phe-Leu-B by the condensation of A-Phe-OH with H-Leu-B have been determined by means of analytical high performance liquid chromatography. Variation of the A group led to large changes in the initial rates of the condensation reaction, and the effect of such changes was found to be similar to that previously found for the secondary specificity of pepsin in the hydrolysis of oligopeptide substrates. Replacement of the Phe and Leu residues of A-Phe-OH or H-Leu-B by other amino acid residues gave relative rates of synthesis in accord with the known primary specificity of the hydrolytic action of pepsin. Partially-acetylated pepsin, which exhibits enhanced hydrolytic activity, also catalyzed the condensation reaction more effectively. The results are discussed in relation to the potential utility and limitations of pepsin as a catalyst in the preparative synthesis of oligopeptides and to the problem of the mechanism of its action.     

THE ACTIVITY OF PARTIAL REACTIONS IN THE CHAIN ELONGATION OF PALMITOYL-CoA AND STEAROYL-CoA BY MOUSE BRAIN MICROSOMES

Partial reactions in the overall chain elongation of palmitoyl-CoA and stearoyl-CoA by mouse brain microsomes have been analyzed. The rate of the initial condensation reaction between palmitoyl-CoA and malonyl-CoA was more than 5 times greater than the rate obtained with stearoyl-CoA, and in both cases good agreement between condensation and overall chain elongation rates was observed. By contrast, both β-hydroxyoctadecanoyl-CoA and β-hydroxyeicosanoyl-CoA were quite rapidly dehydrated by brain microsomes at similar rates. Similar results were obtained with 2-trans-octadecenoyl-CoA and 2-trans-eicosenoyl-CoA in which both substrates were rapidly reduced at nearly the same rate in the presence of NADPH. In all cases, intermediate reactions subsequent to condensation were much more rapid than overall chain elongation. These results suggest that the mechanism of malonyl-CoA-dependent fatty acid chain elongation in brain microsomes is similar to that observed in other tissues, and are consistent with an overall regulation of chain elongation mediated primarily by the initial condensation reaction.     

Synthetic Studies on Dehydrorotenone

The condensation of acetylsalicylchloride with the morpholine enamine of cyclohexanone in the presence of triethylamine gave an adduct which was converted into tetrahydroxanthone (X) by treating with aqueous pyridine containing piperidine. The reaction was applied to condensation of the pyrrolidine enamine (VI) of 6,7-dimethoxychroman-3-one with acetyl tubaic acid chloride to afford dehydrorotenone (II).     

Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II

We report the development of a new method for producing mitotic extracts from tissue culture cells. These extracts reproducibly promote the condensation of chromatin in vitro when incubated with purified interphase nuclei. This condensation reaction is not species specific, since nuclei from chicken, human, and hamster cell lines all undergo chromatin condensation upon incubation with the extract. We have used this extract to investigate the role of DNA topoisomerase II (topo II) in the chromosome condensation process. Chromatin condensation does not require the presence of soluble topo II in the mitotic extract. However, the extent of formation of discrete chromosome-like structures correlates with the level of endogenous topo II present in the interphase nuclei. Our results further suggest that chromatin condensation in this extract may involve two processes: chromatin compaction and resolution into discrete chromosomes.     

Biosynthesis of itaconic acid by aspergillus terreus

Summary The formation of itaconic, aconitic and other acids from glucose in the presence of some enzyme inhibitors or organic acids by Aspergillus terreus was studied. Moreover, the metabolic activities of the preformed mats when floated on solutions of some organic acids were traced.When the resulting information were collected together a presumed condensation reaction between acetate and succinate could be formulated. The reaction product, presumably 1, 2, 3 propane tricarboxylic acid, would undergo a dehydrogenation reaction to yield aconitic acid and subsequently itaconic acid. It has also been suggested that aconityl CoA may be the metabolic form which suits the reactions leading to the formation of itaconic acid. The presumed aconityl CoA may be formed either through a condensation reaction between acetyl CoA and succinate or acetate with succinyl CoA.     

Catalyst-free tandem aldol condensation/Michael addition of 1,3-cyclohexanediones with enolizable aldehydes

An efficient tandem aldol condensation/Michael addition process of unactivated aldehydes and 1,3-cyclohexanedione is described. This transformation proceeds without any catalyst at room temperature with high isolated yields. By a fine-tuning of reaction conditions an access to both the aldol condensation/Michael addition products or to the dehydrated cyclized 9-substituted 1,8-dioxo-xanthenes is given.