Showing papers on "Substrate (chemistry) published in 1974"
TL;DR: It is demonstrated that the yield of a growth process may be accurately computed by considering the relevant biochemistry of conversion reactions and the cytological implications of biosynthesis and growth.
915 citations
TL;DR: The synthesis and analytical use of succinyl-( l -alanine) 3 - p -nitroanilide, a new elastase substrate that is virtually not attacked by trypsin or chymotrypsin and does not undergo appreciable spontaneous hydrolysis is described.
709 citations
01 Jan 1974
TL;DR: This chapter discusses about lactate dehydrogenase (LDH), which was first crystallized from rat muscle in 1943, which is applied in biochemistry and clinical chemistry and is not linked to the pyridine nucleotide coenzymes.
Abstract: Publisher Summary
This chapter discusses about lactate dehydrogenase (LDH), which was first crystallized from rat muscle in 1943. LDHs from bacteria and yeast are not linked to the pyridine nucleotide coenzymes. The LDH activity in serum results from the presence of different enzyme proteins with the same action and substrate specificity, but of different origin. LDH is applied in biochemistry and clinical chemistry. The optimum conditions for the measurements of the enzyme in serum after myocardial infarction, in liver damage, blood diseases, and tumors have been more or less established. The sodium pyruvate content should be at least 99%. Deterioration of the buffer/substrate solution is usually because of bacterial contamination, which can be prevented by addition of a few drops of chloroform. Sudhof et al. have measured the loss of activity on storage of serum at various temperatures. With highly active serum a large proportion of the NADH can be oxidized between the addition of the serum and the first reading. LDH is a cytoplasmic enzyme; simple homogenization in a Potter–Elvehjem homogenizer is sufficient to completely extract the entire enzyme.
598 citations
TL;DR: The decrease in HSP70 following heat shock at 25°C raises the possibility that changes in the protein profile at this temperature may not be related to a heat shock response, even though 25% for 4.5 hours has been shown to confer increased thermotolerance.
Abstract: Dormant grape (Vitis vinifera L.) cuttings were removed from cold storage (3°C) and incubated at 25°C or 45°C for 30 minutes (heat shock treatment), followed by four hours at 25°C. Control cuttings were held at 3°C. Bud proteins were separated by two-dimensional electrophoresis. Thirty-two proteins accumulated following heat shock at 25°C, and 112 accumulated following heat shock at 45°C. Relative to the control, 10 proteins decreased in the 25°C heat shocked buds, but were present at similar or greater concentrations in the 45°C heat shocked buds. Two-dimensional western immunoblots revealed three HSP70 isoforms (pIs 5.84, 5.86, and 5.90) in the control buds, and much larger amounts of two of the isoforms (pIs 5.86, and 5.90) in the 45°C heat shocked buds. The 25°C heat shocked buds had a small amount of only one HSP70 isoform (pI 5.90). The decrease in HSP70 following heat shock at 25°C raises the possibility that changes in the protein profile at this temperature may not be related to a heat shock response, even though 25°C for 4.5 hours has been shown to confer increased thermotolerance. Heat shock at 45°C, which confers greater thermotolerance, is correlated with increased expression of HSP70 as well as other proteins.
535 citations
TL;DR: In this article, a variety of pure and complex cellulosic materials were hydrolyzed by culture filtrates, and the rate and extent of hydrolysis was controlled by degree of crystallinity, particle size, and presence of impurities.
Abstract: Waste cellulose was a suitable carbon source for cellulose production by Trichoderma viride. The enzyme can be produced in submerged fermentation using newspaper as a growth substrate. A variety of pure and complex cellulosic materials were hydrolyzed by culture filtrates. Saccharification of 5% slurries after 48 hr ranged from 2–92%. The rate and extent of hydrolysis was controlled by degree of crystallinity, particle size, and presence of impurities. Newspaper was used to evaluate methods for the pretreatment of substrate. The best pretreatment was ball milling which gave good size reduction, maximum bulk density, and maximum susceptibility. Hammer milling, fluid energy milling, colloid milling, or alkali treatments were less satisfactory. Dissolving cellulose in cuprammonium, or carbon disulfide (Viscose) and then reprecipitating gave a susceptible, but low bulk density product. However the susceptibility was lost if the substrate was dried. Because of costs, low bulk density, necessity of keep ing the substrate wet, and generation of chemical waste streams dissolving cellulose to increase reactivity does not seem a practical approach.
Cellulose fractions separated from municipal trash or agricultural residues such as milled fibres from bovine manure are promising substrates for conversion.
445 citations
TL;DR: In this article, the optical effect of the substrate on the anomalous absorption of aggregated silver films is taken into account in terms of the dipole interaction between an island particle and its mirror image in the substrate.
326 citations
TL;DR: The effects of hydrogen-ion concentration on enzyme activity are consistent with the view that unprotonated amines are the species which bind to the enzyme, and led to formulation of a proposed mechanism of dehydrogenation which does not depend upon intermediate formation of Schiff base from the substrate amine.
318 citations
TL;DR: Initial velocity, product inhibition, and binding studies indicate a biphasic kinetic mechanism in which the reaction pathway depends on the concentration of the substrates, and a numerical rate equation was developed which describes initial velocities over the entire range of substrate concentrations.
264 citations
TL;DR: Initial velocity studies with the dithiothreitol-activated enzyme are compatible with an ordered reaction catalyzed by ribulose 1,5-diphosphate carboxylase, which is immunologically unusual in that antibodies do not form precipitates in immunodiffusion with the enzyme from any source outside of the Athiorhodaceae.
194 citations
TL;DR: The function deduced for a-lactalbumin in the lactose synthetase system is discussed in relation to its structure and a procedure is indicated for quantitatively measuring activity differences in a- lactalbumins.
Abstract: Reactions catalyzed by human milk N-acetyl lactosamine synthetase in the presence and absence of a-lactalbumins have been investigated by steady-state kinetics. N-Acetyl lactosamine synthesis and lactose synthesis in the absence of a-lactalbumin appear to proceed by an ordered sequential reaction, with substrates attaching in the order : Mn2+, UDP-galactose and monosaccharide. Under the conditions used (pH 7.4, 37 “C) the attachment of Mn2+ is not at thermodynamic equilibrium and it appears that the enzyme can accept either free UDP-galactose or its Mn2+ complex as substrate. Evidence is presented which suggests that the Mn2+ complex of UDP may be the final product released from the enzyme. Reactions in the presence of a-lactalbumin proceed by a similar ordered mechanism. Kinetic effects observed in the presence of human a-lactalbumin with three different monosaccharide acceptors, and in the presence of bovine a-lactalbumin with glucose, can be reasonably explained only by assuming that a-lactalbumin attaches to the enzyme immediately before monosaccharides, contrary to suggestions by other workers. It is proposed that a-lactalbumin attaches to an enzyme . Mn2+ . UDPgalactose complex at thermodynamic equilibrium, producing a new enzyme form with increased affinity for monosaccharides. The inhibitory effects of a-lactalbumin on N-acetyl lactosamine synthesis are attributed to inhibition resulting from attachment of the protein to a central complex in an alternative pathway in the reaction scheme. The kinetic effects of four a-lactalbumins with human galactosyl transferase are characterized and intrinsic differences shown to be independent of the source of galactosyl transferase. The function deduced for a-lactalbumin in the lactose synthetase system is discussed in relation to its structure and a procedure is indicated for quantitatively measuring activity differences in a-lactalbumins.
183 citations
TL;DR: A new concept, that of a mnemonical transition, is proposed to explain the departure from Michaelian behavior of monomeric enzymes following ordered reaction mechanisms.
Abstract: A new concept, that of a mnemonical transition, is proposed to explain the departure from Michaelian behavior of monomeric enzymes following ordered reaction mechanisms. The concept integrates three simple ideas: the free enzyme occurs under two conformational states in equilibrium; the collision of any of these forms with the first substrate induces the same third new configuration required for proper substrate binding; the collision of only one of these enzyme forms with the last product stabilizes that form without any new conformational change. This whole set of definitions is equivalent to assuming that the free enzyme which is released after catalysis, is in a conformation different from the initial one. The enzyme can be said to “recall” for a while the configuration stabilized by the last product before relapsing to the initial conformation. The non-hyperbolic behavior is thus the consequence of the cooperation of two different conformations of the free enzyme to the overall reaction process.
The reciprocal steady-state rate equations have been established and thoroughly discussed both for one-substrate, one-product and two-substrate, two-product mnemonical enzymes. The departure from Michaelian behavior does not appear as a consequence of a slow conformational transition, but is defined in a simpler way by the relative values of the activation free energies of conformation changes required for substrate binding on the two enzyme forms.
A two-substrate, two-product enzyme following an ordered reaction mechanism and exhibiting the mnemonical transition has a very distinctive kinetic behavior. The curvature of the primary plots is observed with regard to the first substrate only, and is independant of the concentration of the second substrate as well as that of the first product. The enzyme is not inhibited by an excess of the substrate and the primary plots are either concave up or down. The slopes and the intercepts of the straight lines obtained in double reciprocal plots with the second substrate should give, when these are replotted against the reciprocal of the first substrate concentration, a straight line and a curve, respectively. The cooperation of the enzyme conformations to the overall reaction process can be either positive or negative. Since the reciprocal plots cannot exhibit an extreme, the extent of that cooperation can be measured by the numerical value of the second derivative of the reciprocal rate equation. The extent of cooperation between the free enzyme forms is highly controlled by the concentration of the last product. If the cooperation was already negative, the product strengthens that cooperation. If, on the other hand, the cooperation was positive, the product decreases or even reverses that cooperation.
A very general property of one-sited mnemonical enzymes is that cooperation between enzyme forms is only kinetic and does not appear in the substrate binding isotherms.
TL;DR: The hydroxylation activity is stable to dialysis but decreases to about 20 to 25% of the original level when the 100,000 x g supernatant is passed through Sephadex G-25, presumably due to the loss of a small protein component.
TL;DR: A basic, progesterone-induced, intrauterine glycoprotein with a purple coloration can be purified from the uterine fluids of ovariectomized female pigs given daily doses of progester one by successive chromatography on columns of CM-cellulose and Sephadex G-100.
TL;DR: It is concluded that aortic lysyl oxidase is a copper metalloenzyme that catalyzed the formation of tritiated water from embryonic aortas grown in organ culture.
TL;DR: Chloride binding induces a subtle conformational change in the enzyme reflected by the suppression of the exchange of 26 protons and a 240-fold increase in the amylase-Ca2+ binding constant from 8.3 × 108 to 2 × 1011 M−1.
Abstract: α-Amylase from hog pancreas is shown to possess one binding site for Cl− per molecule of enzyme with a dissociation constant of 3 × 10−4 at 25 °C. The chloride anion functions as an activating effector increasing kcat 30-fold towards either starch or p-nitrophenylmaltoside. No change in Km for either substrate is seen. The other monovalent anions Br−, I−, NO2−, NO3−, ClO4−, SCN−, N3− and CNO− can substitute for chloride but their effect on kcat decreases as their anionic radius increases. Chloride binding induces a subtle conformational change in the enzyme reflected by the suppression of the exchange of 26 protons and a 240-fold increase in the amylase-Ca2+ binding constant from 8.3 × 108 to 2 × 1011 M−1. The conformational change is minor since it is not detected by circular dichroism, protein fluorescence or by specific probes attached to the enzyme. The ability of small structural changes to induce large catalytic acceleration is discussed.
TL;DR: Electronic mechanisms of reactions correlate the appearance and the disappearance of the various enzyme forms with known intermediates of indole-3-acetate degradation, and are associated with the formation of 3-methyleneoxindole as the major end-product of indoles3- acetate degradation.
Abstract: A study of reaction mechanisms of indole-3-acetate degradation by various peroxidases, in the absence of added hydrogen peroxide, has been performed employing stopped-flow and low-temperature spectroscopic techniques.
At pH 6 and for high [substrate]/[enzyme] ratios, peroxidase compound II is formed and is stable for several minutes. However, this enzyme compound becomes visible only after the transient appearance of ferroperoxidase and of compound III. This reaction process is apparently linked with the formation of 3-methyleneoxindole as the major end-product of indole-3-acetate degradation.
On the other hand, when indole-3-acetate and peroxidase in stoichiometric concentrations are mixed at pH 4, only ferroperoxidase and compound III are formed and the process appears to be linked with the production of indole-3-aldehyde.
Stopped-flow experiments show that indole-3-acetate is rapidly bound on peroxidase, this rapid binding step being followed by a slow one-electron transfer between indole-3-acetate and peroxidase. This reduction of ferriperoxidase is blocked at low temperature (–20 °C), whereas in these experimental conditions, peroxidation of indole-3-acetate in the presence of added hydrogen peroxide still occurs. Moreover, when compound II reacts with indole-3-acetate at low temperature (–15 °C), this compound is regenerated as long as oxygen is present in the reaction medium. Neither ferroperoxidase nor compound I appear during this process.
Most, if not all, of the compound III is formed by oxygenation of the ferrous peroxidase. This compound III can react with an equimolar amount of indole-3-acetate to give ferrous peroxidase and indole-3-aldehyde. Moreover, compound III can react with two indole-3-acetate molecules leading to compound II formation. Because only small amounts of the organic peroxide derived from indole-3-acetate are present in the reaction medium, most of the compound II is formed by a two-equivalent reduction of compound III, rather than through the direct interaction of the organic peroxide with peroxidase.
Electronic mechanisms of reactions, consistent with the present experimental data, are proposed and discussed. They correlate the appearance and the disappearance of the various enzyme forms with known intermediates of indole-3-acetate degradation.
TL;DR: The rationale of the method to be described for establishing the vectorial arrangement of membrane proteins is based on the ability of the enzyme lactoperoxidase to catalyze iodination of tyrosine and histidine residues of proteins.
Abstract: Publisher Summary The rationale of the method to be described for establishing the vectorial arrangement of membrane proteins is based on the ability of the enzyme lactoperoxidase to catalyze iodination of tyrosine and histidine residues of proteins. Studies of the reaction catalyzed by lactoperoxidase have shown that the enzyme forms an enzyme-substrate complex with the substrate which is halogenated. In the case of proteins, this would be the phenolic group of tyrosine or the imidazole group of histidine. The enzyme is a high molecular weight protein (77,500) that does not dissociate into subunits, and therefore does not readily pass through an intact membrane. Thus, in the case of an intact membrane system, only those proteins which are on the surface of the membrane exposed to the enzyme and which could form an enzyme-substrate complex with the lactoperoxidase will be iodinated. In contrast, when the organization of a membrane has been disrupted and the enzyme has access to both surfaces of the membrane, those proteins on both surfaces which are accessible to the enzyme would be iodinated; and finally, when the membrane components are totally dissociated, all membrane components would be accessible to the enzyme and should be iodinated.
TL;DR: A simple and sensitive one-step chemical assay using dual-wavelength spectrophotometry for dopamine-β-hydroxylase (DBH) activity is described, permits the assay of the maximum velocity at a saturated concentration of substrate under the optimum conditions and is generally applicable to various tissues with low enzyme activity.
TL;DR: The apoenzyme obtained on removal of Zn(2+) from beta-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn (2+)-containing enzyme, which raised the question of whether the thiol group in the enzyme is a metal-ion ligand.
Abstract: 1. The apoenzyme obtained on removal of Zn2+ from β-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn2+-containing enzyme. 2. Removal of Zn2+ led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn2+ by Co2+, Cd2+, Mn2+ or Hg2+ gave enzymes with significant, but lower, β-lactamase activity. No activity was detected in the presence of Cu2+, Ni2+, Mg2+ or Ca2+. 4. Equilibrium dialysis indicated that the enzyme had at least two Zn2+ binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn2+ indicated that activity paralleled binding of Zn2+ to the site of highest affinity. 5. Replacement of Zn2+ by Co2+ and Cd2+ gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured β-lactamase II with Ellman's reagent [5,5′-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce β-lactamase II activity in high yield.
TL;DR: A method for measuring γ-glutamyltransferase (EC 2.3.2.2) activity in serum, which can be used with automated enzyme analyzers that require enzyme reactions to be initiated with substrate, is described.
Abstract: We describe a method for measuring γ-glutamyltransferase (EC 2.3.2.2) activity in serum, which can be used with automated enzyme analyzers (such as the LKB 8600 Reaction Rate Analyzer) that require enzyme reactions to be initiated with substrate. The method also permits optimal determination conditions to be obtained at 37 °C. The enzymatic reaction is commenced by adding γ-glutamyl- p -nitroanilide dissolved in dilute hydrochloric acid to samples pre-incubated with tris(hydroxymethyl)aminomethane—glycylglycine buffer. The p -nitroaniline liberated is continously monitored at 37 °C at 405 nm. The pH of the pre-incubation buffer is such that the optimal pH for the enzyme reaction results from addition of the acid substrate solution.
TL;DR: Results of denaturation studies showed that beta-1,4-glucanase and chitosanase activities decreased at equal rates, and Cellotriose was the smallest polymer capable of hydrolysis.
Abstract: An enzyme that has both beta-1,4-glucanase and chitosanase activities is characterized. Evidence for homogeneity was obtained from electrophoresis and sedimentation velocity studies; only one N-terminal amino acid, valine, was found. Results of denaturation studies showed that beta-1,4-glucanase and chitosanase activities decreased at equal rates. With carboxymethylcellulose as the substrate, a K(m) of 1.68 g of carboxymethylcellulose per liter of solution and a V(max) of 2.20 x 10(-9) mol/min were found. With chitosan (the beta-1,4-polymer of glucosamine) as the substrate, a K(m) of 0.30 g of chitosan per liter of solution and a V(max) of 0.75 x 10(-9) mol/min were found. A pH optimum of 5.0 was found for beta-1,4-glucanase activity, and pH optima of 5.0 and 6.8 were found for chitosanase activity. beta-1,4-Glucanase activity had a temperature optimum of 38 C, and chitosanase activity had a temperature optimum of 70 C. Chitosan stabilized both enzyme activities at 70 C. Cellotriose was the smallest polymer capable of hydrolysis. Glucosamine was released by action of the enzyme upon cell wall preparations of several fungi.
TL;DR: Maximal efficiency of catalysis appears to require that an enzyme and a substrate combine productively in the ground state, and catalytic complexes must be capable of rapid decomposition by two opposite routes which are not related by symmetry.
Abstract: Maximal efficiency of catalysis appears to require that an enzyme and a substrate combine productively in the ground state. Following this initial combination, powerful forces of attraction are believed to develop between the enzyme and activated forms of the substrate. Local conformation changes may assist this process, if the active site of the enzyme is initially open to substrate access, but tends toenclose the altered substrate in the transition state. In many cases, catalytic complexes must be capable of rapid decomposition by two opposite routes which are not related by symmetry. Structural reorganization of the active site may help to satisfy this additional requirement.
TL;DR: It is concluded that the two peroxidases are kinetically different with distinct substrate profiles and that they may fulfill different physiological functions.
TL;DR: The substrate specificity of the purified enzyme appears to be unique, since it hydrolyzed only ovalbumin glyco-peptides among several glycopeptides and their derivatives.
TL;DR: In this paper, it was shown that the ureido-N position of biotin is the site of carboxylation, rather than the UREido-O, of the imidazolidone ring.
TL;DR: The glucose-6-P-dependent form of glycogen synthetase has been purified about 5,000-fold from yeast extracts and shows a single band on disc gel electrophoresis and possesses a molecular weight of 300,000, as measured by sedimentation equilibrium.
TL;DR: The proposed mechanism predicts the existence of a stable enzyme-NAD+ complex, and this form is well known, and agrees with many of the published observations on the enzyme but differs from those indicated by previous kinetic studies.
TL;DR: A theoretical treatment of the kinetics of reactions catalyzed by enzymes attached to the inner surface of a tube, through which the substrate solution passes, and criteria for insignificant and significant diffusional effects are proposed.
Abstract: A theoretical treatment is given of the kinetics of reactions catalyzed by enzymes attached to the inner surface of a tube, through which the substrate solution passes. A utilization factor, the ratio of the actual reaction rate to that in the absence of diffusional effects, is defined. A numerical procedure is proposed and numerical and approximate solutions for the utilization factor are given for five kinetic conditions: (a) Michaelis-Menten behavior, (b) substrate inhibition, (c) product inhibition (competitive), (d) product, inhibition (non-competitive), and (e) product inhibition (anticompetitive). When the enzyme chemically attached to a tube obeys a Michaelis-Menten relationship, criteria for insignificant and significant diffusional effects are proposed.