Showing papers on "Substrate (chemistry) published in 1983"
TL;DR: An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules of rat anterior, intermediate, and neural pituitaries and bovine intermediate pituitary.
Abstract: An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules of rat anterior, intermediate, and neural pituitary and bovine intermediate pituitary. High levels of endogenous inhibitors of this alpha-amidation activity have also been found in tissue homogenates. The alpha-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, and EDTA; alpha-amidation activity is restored to above control levels upon addition of copper. The alpha-amidation reaction requires the presence of molecular oxygen. Of the various cofactors tested, ascorbic acid was the most potent stimulator of alpha-amidation. The alpha-amidation activity has a neutral pH optimum and is primarily soluble following several cycles of freezing and thawing. Kinetic studies with the bovine intermediate pituitary granule-associated activity demonstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was the varied substrate; the apparent Km and Vmax varied with the concentration of ascorbic acid. The substrate specificity of the alpha-amidation activity appears to be quite broad; the conversion of D-Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a variety of glycine-extended peptides.
344 citations
TL;DR: A type IV collagen-degrading enzyme activity secreted by a highly metastatic mouse tumor was purified by concanavalin A- and typeIV collagen-agarose affinity chromatographies followed by gel filtration on Bio-Gel A-0.5 m, indicating that the enzyme is a hydrophobic protein.
247 citations
TL;DR: In this paper, it was shown that the ferric reducing activity in the roots of iron-deficient bean plants is located in an enzyme in the plasmalemma of the cortex or epidermis cells.
Abstract: Iron-deficient bean plants (Phaseolus vulgaris L. cv. Prelude) exhibited a ferric reducing activity in the roots, with kinetics characteristic for matrix-bound enzymes: the reaction rate was proportional to substrate (Fe-EDTA) concentration until 100 μM, and at higher concentrations it leveled off to a maximum; the Lineweaver-Burk plot yielded a non-linear relationship between rate −1 and substrate −1. The Arrhenius plot yielded apparent activation energies which were dependent on substrate concentration. No evidence was obtained for the secretion by roots of a low molecular weight metabolite involved in the reduction of iron prior to its uptake. The results are interpreted to indicate that the ferric reducing activity in the roots of iron-deficient bean plants is located in an enzyme in the plasmalemma of the cortex or epidermis cells.
220 citations
Patent•
05 Oct 1983
TL;DR: In this paper, a surface-modified electrodes which may be used in electrochemical cells for production of electrical energy comprise an enzyme immobilized on a support consisting of at least a monolayer coating of a carbonaceous pyropolymer possessing recurring units containing at least carbon and hydrogen atoms composited on a high surface area refractory inorganic oxide.
Abstract: Surface-modified electrodes which may be used in electrochemical cells for production of electrical energy comprise an enzyme immobilized on a support. The support consists of at least a monolayer coating of a carbonaceous pyropolymer possessing recurring units containing at least carbon and hydrogen atoms composited on a high surface area refractory inorganic oxide such that the carbonaceous pyropolymer monolayer coating replicates the surface area and macropore volume of the inorganic oxide. The coated support is then treated by impregnation with a water-soluble polyamine followed by contact with a solution of a molar excess of a bifunctional monomeric material to form a copolymer which provides pendant bonding sites. The copolymer is entrapped and adsorbed in the pores of the support material to provide a permanent attachment thereto. The treated support is then contacted with an excess of an enzyme to effect the conjugate attachment of the enzyme to the treated support. The immobilized enzyme will act as a working electrode in the presence of a predetermined substrate such as glucose to provide electrical energy.
172 citations
TL;DR: Methods for determination involve digestion of natural fats of known structure and synthetic acylglycerols followed by analysis of the lipolysis products and applications of these procedures are discussed and data on the specificity of various lipases presented is presented.
Abstract: Specificity of lipases is controlled by the molecular properties of the enzyme, structure of the substrate and factors affecting binding of the enzyme to the substrate. Types of specificity are as follows. I. Substrate: (a) different rates of lipolysis of TG, DG, and MG by the same enzyme; (b) separate enzymes from the same source for TG, DG and MG. II. Positional: (a) primary esters; (b) secondary esters; and (c) all three esters or nonspecific hydrolysis. III. Fatty acid, preference for similar fatty acids. IV. Stereospecificity: faster hydrolysis of one primary sn ester as compared to the other. V. Combinations of I-IV. Lipases with these specificities are: Ia, pancreatic; Ib, postheparin plasma. IIa, pancreatic; IIb, Geotrichum candidum, for fatty acids with cis-9-unsaturation, and IIc, Candida cylindracea. III, G. candidum for unsaturates. IV. sn-1, postheparin plasma and sn-3 human and rat lingual lipases. V. Rat lingual lipase. Methods for determination involve digestion of natural fats of known structure and synthetic acylglycerols followed by analysis of the lipolysis products. All of the types of specificity have been detected with use of synthetic acylglycerols. Detection of stereospecificity requires enantiomeric acylglycerols which are difficult to synthesize, so other methods have been developed. These involve the generation of 1,2-(2,3)DG and resolution of the enantiomers. Trioleoylglycerol or racemic TG can be used as substrates. If the lipase is stereospecific, then either the sn-1,2- or 2,3-enantiomer will predominate. The relative amounts of the enantiomers can be determined by measurement of specific rotation, and nuclear magnetic resonance spectra. The DG can also be separated by conversion to phospholipids and hydrolysis with phospholipases A-2 or C. Applications of these procedures are discussed and data on the specificity of various lipases presented.
130 citations
TL;DR: Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration.
Abstract: Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The Km value was found to be 0.48 × 10−4 M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels.
119 citations
112 citations
TL;DR: The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at acidic pH 9.0.
Abstract: The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.
105 citations
TL;DR: Experiments performed with the partially purified and separated forms confirmed that p-nitrophenol, phenol, 6-OH-melatonin and acetaminophen were substrates for both forms of platelet PST.
Abstract: Human platelets contain at least two forms of phenol sulfotransferase (PST), a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which micromolar concentrations of phenol can serve as substrate. At higher concentrations phenol is also a substrate for the TL form. Studies of the regulation and the possible clinical value of measurements of platelet PST have been hampered because there is no specific substrate for the TS form of the enzyme. The purposes of these experiments were to determine whether there might be a better substrate than phenol for use in measurement of the activity of the TS form of platelet PST, and to attempt to physically separate the two forms of the platelet enzyme. The results of substrate kinetic, thermal stability, and inhibitor studies performed with platelet homogenates were all compatible with the conclusion that p-nitrophenol and 6-OH-melatonin were substrates for both the TS and TL forms of platelet PST. Norepinephrine, epinephrine and 5-OH-tryptamine were substrates for only the TL form. The apparent K
m constants of the two forms of PST for p-nitrophenol differed by 7,100-fold when measured in platelet homogenates. This difference was 200 times greater than that which has been reported for phenol. Therefore, p-nitrophenol is the preferred substrate for measurement of the TS PST activity if interference by the TL activity is to be avoided. This information made it possible to use p-nitrophenol as a substrate in experiments designed to separate the two forms of platelet PST. The TS and TL forms of the platelet enzyme were separated by ion exchange chromatography. Experiments performed with the partially purified and separated forms confirmed that p-nitrophenol, phenol, 6-OH-melatonin and acetaminophen were substrates for both forms. Apparent K
m constants of the two forms differed most for p-nitrophenol.
104 citations
TL;DR: The alpha-amylase produced by Bacillus licheniformis CUMC305 was purified with a 42% yield through a series of four steps as discussed by the authors, and the enzyme showed maximal activity at 90 degrees C and pH 9.0, and 91% of this activity remained at 100 degrees C. The activation energy for this enzyme was calculated as 5.1 x 10 J/mol.
Abstract: alpha-Amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90 degrees C and pH 9.0, and 91% of this activity remained at 100 degrees C. The enzyme retained 91, 79, and 71% maximal activity after 3 h of treatment at 60 degrees C, 3 h at 70 degrees C, and 90 min at 80 degrees C, respectively, in the absence of substrate. On the contrary, in the presence of substrate (soluble starch), the alpha-amylase enzyme was fully stable after a 4-h incubation at 100 degrees C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74, 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 h of treatment. The activation energy for this enzyme was calculated as 5.1 x 10 J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. V(max) values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na, Ca, and Mg, showed stimulatory effect, whereas Hg, Cu, Ni, Zn, Ag, Fe, Co, Cd, Al, and Mn were inhibitory. Of the anions, azide, F, SO(3), SO(4), S(2)O(3), MoO(4), and Wo(4) showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, beta-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. alpha-Amylase was fairly resistant to EDTA treatment at 30 degrees C, but heating at 90 degrees C in presence of EDTA resulted in the complete loss of enzyme activity, which could be recovered partially by the addition of Cu and Fe but not by the addition of Ca or any other divalent ions.
99 citations
TL;DR: Variations of substrate transport across the cerebral capillary endothelium were examined in response to variations of the substrate demand of the brain tissue, and to variations in the blood concentration of glucose and ketone bodies.
Abstract: Variations of substrate transport across the cerebral capillary endothelium were examined in response to variations of the substrate demand of the brain tissue, and to variations of substrate concentration in the blood. The substrates examined included glucose and ketone bodies. The transport changes were measured in rats, using an indicator fractionation method modified by the reviewer.
Four mechanisms appeared to contribute to the adjustment of substrate transport to variations in substrate demand. The first and least important mechanism was the change of concentration gradient across the endothelium that occurred when the substrate consumption rate changed. The second mechanism was the flow-dependency of the average capillary substrate concentration: the higher the perfusion rate, the higher the average capillary concentration. This mechanism failed to account for the changes of substrate transport observed during marked increases of the metabolic rate.
The third and most important mechanism was a change of the capillary diffusion capacity, probably associated with a change of the number of perfused capillaries.
The fourth mechanism, not previously described, was an adaptation of transport to permanent changes of substrate concentration in the blood. This mechanism appeared to reflect changes of the concentration (and affinity?) of transport proteins in the plasma membranes of endothelial cells, possibly in association with changes of cellular protein synthesis and gene expression.
TL;DR: Propranolol bound to membranes may prevent cytoplasmic enzyme action, probably by decreasing the availability of substrate through the formation of stable lipid-drug-protein complexes.
Abstract: Propranolol, a cationic amphiphilic drug, caused enhanced incorporation of labeled precursor into phosphatidic acid and its metabolites in rat cerebral cortex mince, suggesting increased biosynthesis or reduced degradation. Inhibition of phosphatidate phosphohydrolase could explain the observed drug-induced accumulation of phosphatidic acid and other acidic lipids. Propranolol exhibited differential effects on the free and membrane-bound forms of phosphatidate phosphohydrolase. The drug inhibited cytoplasmic enzyme in a dose-dependent manner only when membrane-bound substrate was used but had practically no effect on the membrane-bound enzyme irrespective of the nature of the substrate used or on the cytoplasmic enzyme when free substrate was used. Brain cytoplasmic enzyme obtained from rats sacrificed 30 min after intraperitoneal injections of propranolol did not show any inhibition. propranolol bound to membranes may prevent cytoplasmic enzyme action, probably by decreasing the availability of substrate through the formation of stable lipid-drug-protein complexes.
TL;DR: The kinetic mechanism, the amount of chloride required, and the effect of pH on activation, plus the relative activating potencies of various anions, are all strongly dependent on the substrate employed, and possible explanations for this substrate dependence are offered.
Abstract: Anion activation of pulmonary angiotensin converting enzyme has been examined by using 23 furanacryloyl- and 3 benzoyl-tripeptides as substrates. Chloride stimulates hydrolysis of all substrates at least 24-fold. However, the kinetic mechanism, the amount of chloride required, and the effect of pH on activation, plus the relative activating potencies of various anions, are all strongly dependent on the substrate employed. Three substrate classes have been identified. Class I substrates appear to be hydrolyzed at pH 7.5 by an ordered bireactant mechanism in which anion must bind before substrate. The apparent activation constant (KA') for Cl- ranges from 75 to 150 mM at pH 7.5, doubles at pH 9.0, and decreases to about 3 mM at pH 6.0. Class II substrates, in contrast, are hydrolyzed by a nonessential activator mechanism. The kinetically determined KA' for Cl- at pH 7.5 ranges from 2.9 to 5.0 mM and changes only slightly with pH. Class III substrates are also hydrolyzed by a nonessential kinetic mechanism but one different from that followed by class II peptides. KA' values for Cl- at pH 7.5 measured with class III substrates are 18-30 mM. Class II substrates have Arg or Lys at the ultimate or penultimate position. The features distinguishing class I and III peptides are less clear, although all class III substrates identified have penultimate alanine residues. Possible explanations for this substrate dependence are offered.
TL;DR: The experiments showed that the amidating enzyme has a mandatory requirement for glycine in position 3 of the tripeptide substrates; peptides containing lysine, glutamic acid, leucine or alanine in the C-terminal position did not undergo reaction.
TL;DR: The possibility is discussed that chalcone synthase and the corresponding enzyme of fatty acid synthesis in higher plants, beta-ketoacyl-acyl carrier protein synthase, have a common evolutionary origin.
Patent•
08 Feb 1983
TL;DR: A surface modified substrate as mentioned in this paper is a substrate with a complex of a polymeric cationic surfactant and a dialdehyde, which is then adsorbed onto the substrate surface.
Abstract: A surface modified substrate, wherein the substrate carries a complex adsorbed thereto, which complex is of a polymeric cationic surfactant that contains primary amino nitrogens as well as secondary and/or tertiary amino nitrogens, and a dialdehyde that has 1-4 carbon atoms between the two aldehyde groups. To said complex there may additionally be bonded an anionic compound and optionally alternatingly additional cationic and anionic compounds. The modification primarily means that positive or negative charges can be imparted to the original substrate. Secondly, however, the point may be to utilize reactive groups in bonded cationic or anionic compounds. The surface modified substrate is prepared by contacting the substrate with the polymeric cationic surfactant and the dialdehyde at such conditions that a complex is formed, which is adsorbed onto the substrate surface, and optionally continuing the ionic bonding with an anionic compound and possibly also alternatingly additional cationic and anionic compounds. The polymeric cationic surfactant and the dialdehyde are preferably utilized, separately or together, dissolved in a polar solvent, preferably water. The formation of the complex takes place at a pH-value above 8, whereupon the charges are preferably created at a pH-value below 7.
TL;DR: Proposals for the stepwise cleavage of biologically active peptides like substance P and beta-casomorphine by dipeptidyl peptidase IV are proposed.
TL;DR: The proposed method, based on the blue shift of the absorption spectrum that occurs upon hydrolysis of the substrate into furanacryloyl-L-phenylalanine and glycylglycine, is very suitable for routine work, clinical investigation, and kinetic and structural studies of the serum enzyme.
Abstract: The appropriate conditions for determining angiotensin-converting enzyme in human serum with use of 2-furanacryloyl-L-phenylalanylglycylglycine as substrate are described. The method, a modification of that reported by Holmquist et al. (Anal. Biochem. 95:540-548, 1979) for purified rabbit lung enzyme, is based on the blue shift of the absorption spectrum that occurs upon hydrolysis of the substrate into furanacryloyl-L-phenylalanine and glycylglycine. The Km value for the substrate is 0.31 mmol/L. Some kinetic properties determined by this method are similar to those previously reported for the purified serum enzyme. Results by the present assay and Cushman's modified method correlate closely (r = 0.995). The normal reference interval for 42 adult donors was 43-137 U/L (mean 90 U/L, SD 23 U/L). The within-run and between-run CVs ranged from 3.0 to 4.1%. Its rapidity, simplicity, sensitivity, and precision make the proposed method very suitable for routine work, clinical investigation, and kinetic and structural studies of the serum enzyme.
Patent•
06 Apr 1983
TL;DR: In this article, an aqueous dispersion containing water soluble algin to the surface of a food substrate is applied to the align-coated food substrate for a period of time sufficient to form a substantially continuous edible alginate film along the surfaces of the food substrate.
Abstract: A coated food product of this invention is produced by applying an aqueous dispersion containing water soluble algin to the surface of a food substrate. The aqueous dispersion has a viscosity effective to substantially uniformly coat the surface of the food substrate. A dry gelling mixture is then applied to the align-coated food substrate for a period of time sufficient to form a substantially continuous edible alginate film along the surface of the food substrate. The film is sufficient to effectively retard the migration of moisture from the surface of the food substrate thereby retarding dehydration thereof. The film is sufficient to constitute an oxygen barrier for retarding oxidation of the food substrate and to retain flavor within said food substrate. The dry gelling mixture comprises a dry carrier material and a dry gelling agent. The carrier material and gelling agent are in particulate form and have particle sizes sufficient to obtain a substantially uniform gelling mixture.
TL;DR: In this article, the Fourier Transform NMR pulsed-gradient spin-echo technique was used to monitor substrate binding to α- and β-cyclodextrins in solution.
Abstract: It is demonstrated that substrate binding to α- and β-cyclodextrins (CD) in solution can conveniently and directly be monitored from multicomponent self-diffusion data on these solutions, using the Fourier Transform NMR pulsed-gradient spin-echo technique. Included are aromatics and a series of alcohols ranging from methanol to octanol. Experimentally it was found thatn-alcohols associate more strongly with α-CD than with β-CD. As the bulkiness of the alcohol increased, binding to β-CD was enhanced while the reverse effect was observed in the case of α-CD. For both cyclodextrins it was found thatn-alcohol complexation in the homologous series was attributable to an increment in standard free energy of complexation of ∼ −3.0 kJ/mol for each −CH2− group, suggesting that the binding mechanism is of a hydrophobic nature.
TL;DR: Uroporphyrinogen decarboxylase (EC 4.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200.
Abstract: Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an Mr approximately 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of approximately 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol X h-1 X (mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uroporphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions including Fe2+, Co2+, Cu2+, Zn2+, and Pb2+. The pattern of accumulation of intermediate (hepta-, hexa-, and pentacarboxylate porphyrinogens) and final (coproporphyrinogen) decarboxylation products is affected by the ratio of substrate (uroporphyrinogen I or III) concentration to enzyme concentration. Under physiologic conditions where the uroporphyrinogen to enzyme ratio is low, the substrate is nearly quantitatively decarboxylated, and the major product is coproporphyrinogen. If the ratio of uroporphyrinogen to enzyme is high, intermediates accumulate, and heptacarboxylate porphyrinogen becomes the major decarboxylation product.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The excretion of laccase by sycamore cells is significant, being about 2% of the total protein synthesized by the cells during the exponential phase of growth, and is independent of cell growth.
Abstract: A laccase-type polyphenol oxidase is excreted by sycamore cells (Acer pseudoplatanus L.) cells. The enzyme has been purified by classical purification techniques. It is a blue copper protein of Mr 97 000, containing 45% carbohydrate and 0.24% copper. This protein consists of one single unit and the copper content corresponds to four copper atoms per protein molecule. The specific activity of the purified extracellular sycamore-cell laccase measured at pH 6.6 (optimum pH) and in the presence of 20mM-4-methhylcatechol (optimum substrate conditions) corresponded to an oxygen uptake of 32 000 nmol of O2/min per mg of protein. Under these conditions, the catalytic-centre activity of the enzyme reached 100 s-1. The excretion of laccase by sycamore cells is significant, being about 2% of the total protein synthesized by the cells during the exponential phase of growth, and is independent of cell growth. The physiological significance and the problems raised by the passage of this protein across the cytoplasmic membrane are discussed.
TL;DR: It is concluded that 4-pentenoic acid is metabolized in mitochondria by two pathways, and the most effective inhibitor was found to be 3-keto-4-pentenoyl-CoA, which inhibits the enzyme in both a reversible and irreversible manner.
Abstract: The metabolism of 4-pentenoic acid, a hypoglycemic agent and inhibitor of fatty acid oxidation, has been studied in rat heart mitochondria. Confirmed was the conversion of 4-pentenoic acid to 2,4-pentadienoyl coenzyme A (CoA), which either is directly degraded via beta-oxidation or is first reduced in a NADPH-dependent reaction before it is further degraded by beta-oxidation. At pH 6.9, the NADPH-dependent reduction of 2,4-pentadienoyl-CoA proceeds 10 times faster than its degradation by beta-oxidation. At pH 7.8, this ratio is only 2 to 1. The direct beta-oxidation of 2,4-pentadienoyl-CoA leads to the formation of 3-keto-4-pentenoyl-CoA, which is highly reactive and spontaneously converts to another 3-ketoacyl-CoA derivative (compound X). 3-Keto-4-pentenoyl-CoA is a poor substrate of 3-ketoacyl-CoA thiolase (EC 2.3..1.16) whereas compound X is not measurably acted upon by this enzyme. The effects of several metabolites of 4-pentenoic acid on the activity of 3-ketoacyl-CoA thiolase were studied. 3,4-Pentadienoyl-CoA is a weak inhibitor of this enzyme that is protected against the inhibition by acetoacetyl-CoA. The most effective inhibitor of 3-ketoacyl-CoA thiolase was found to be 3-keto-4-pentenoyl-CoA, which inhibits the enzyme in both a reversible and irreversible manner. The reversible inhibition is possibly a consequence of the inhibitor being a poor substrate of 3-ketoacyl-CoA thiolase. It is concluded that 4-pentenoic acid is metabolized in mitochondria by two pathways. The minor yields 3-keto-4-pentenoyl-CoA, which acts both as a reversible and as a irreversible inhibitor of 3-ketoacyl-CoA thiolase and consequently of fatty acid oxidation.
TL;DR: The kinetics of the hydrolysis of fluorsceindiacetate and fluorescein-di-beta-D-galactopyranoside were investigated by thin-layer chromatography and the mathematical model takes into account the competition of substrate and intermediate and the accumulation of the intermediate at the enzyme.
TL;DR: The results indicate that enzyme reduction is rate-limiting when the alcohol form (PNP) is the substrate, but in the case of the amine (PMP), the rate- Limiting step in catalysis occurs subsequent to reduction.
TL;DR: The substrate specificity of beef heart phosphodiesters activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP.
Abstract: The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3',5'-monophosphate (cAMP). Several analogs have increased Km values, but unaltered V values if compared to cAMP; also the contrary (unaltered Km and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP. cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme. The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.