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Showing papers on "Substrate (chemistry) published in 1982"


Journal ArticleDOI
TL;DR: It appears that some mechanism of association with the humic polymer offers the best form of protection, yet permits the retention of enzyme activity, and the mere adsorption of enzymes to soil surfaces does not guarantee subsequent activity.
Abstract: The activity of any particular enzyme in soil is a composite of activities associated with various biotic and abiotic components, e.g. proliferating cells, latent cells, cell debris, clay minerals, humic colloids and the soil aqueous phase. The location of the enzyme is at least partially determined by such factors as the size and solubility of its substrate, the species of microorganism, and the physical and chemical nature of the soil colloids. However, enzymes may change location with time, for example, many hydrolases are intracellular sensu stricto but are also found associated with cell debris and clay and organic colloids. There are difficulties in quantifying the various activities, but this may be possible by employing different types of assays, the prudent use of controls and the study of crude enzyme extracts from soil. Enzymes bound to clay and humic colloids (the immobilized or accumulated enzyme fraction) have a residual activity not found in enzymes free in the soil aqueous phase. However, the mere adsorption of enzymes to soil surfaces does not guarantee subsequent activity, and it appears that some mechanism of association with the humic polymer offers the best form of protection, yet permits the retention of enzyme activity. The catalytic activity of extracellular enzymes is discussed and a possible relationship between soil microorganisms, exogenous substrates and immobilized enzymes is suggested.

966 citations


Journal ArticleDOI
21 Oct 1982-Nature
TL;DR: A general method for systematically replacing amino acids in an enzyme is described, which allows analysis of their molecular roles in substrate binding or catalysis and could eventually lead to the engineering of new enzymatic activities.
Abstract: We describe here a general method for systematically replacing amino acids in an enzyme. This allows analysis of their molecular roles in substrate binding or catalysis and could eventually lead to the engineering of new enzymatic activities. The gene encoding the enzyme is first cloned into a vector from which the enzyme is expressed and is then mutated in vitro to change a particular nucleotide and hence the amino acid sequence of the enzyme. We have cloned the gene for the tyrosyl tRNA synthetase of Bacillus stearothermophilus into a vector derived from the single-stranded bacteriophage M13 to facilitate mutagenesis with mismatched synthetic oligodeoxynucleotide primers. From the recombinant M13 clone we have obtained high levels of the enzyme (∼50% of soluble protein) expressed in the Escherichia coli host and have converted cysteine (Cys35) at the enzyme's active site to serine. This leads to a reduction in enzymatic activity that is largely attributable to a lower Km for ATP.

326 citations


Journal ArticleDOI
TL;DR: The kinetics of benzoyltyrosine ester hydrolysis during the course of irreversible inhibition of the enzyme has been found to be in satisfactory agreement with equations obtained previously and the apparent rate constant between the enzyme and an irreversible inhibitor can be easily obtained in one single experiment by following the Course of substrate hydrolyses in the presence of the inhibitor.
Abstract: On the basis of the equations derived previously [Tsou, C. L. (1965) Sheng Wu Hua Hsueh Yu Sheng Wu Wu Li Hsueh Pao 5, 398-408, 409-417] for the substrate reaction during the course of enzyme modification, the kinetic behavior of the system chymotrypsin-substrate-modifier has been studied. The kinetics of benzoyltyrosine ester hydrolysis during the course of irreversible inhibition of the enzyme has been found to be in satisfactory agreement with equations obtained previously. The apparent rate constant between the enzyme and an irreversible inhibitor can be easily obtained in one single experiment by following the course of substrate hydrolysis in the presence of the inhibitor. The results are also in accord with the assumption that diisopropyl fluorophosphate can be classified as an irreversible competitive inhibitor. For both phenylmethanesulfonyl fluoride and L-1-[(p-toluene-sulfonyl)amino]-2-phenylethyl chloromethyl ketone, the inhibition has been found to be in agreement with the kinetics of the complexing type; i.e., a noncovalent enzyme-inhibitor complex is formed before irreversible enzyme modification. Both the equilibrium constants for the complex formation and the first-order rate constants for the irreversible modification step have been determined also by following the course of substrate hydrolysis in the presence of the irreversible inhibitor.

306 citations


Journal ArticleDOI
TL;DR: In this paper, a kinetic model describing the catalytic activity of redox polymer film electrodes is presented and discussed for reaction schemes in which the primary reaction between the active form of the catalyst and the substrate is the rate-determining step.

306 citations


Journal ArticleDOI
TL;DR: This method of substrate formation using the cholate dialysis technique permits the preparation of large amounts of stable, efficient, homogeneous, and well-defined substrate that is suitable for measuring low levels of enzyme activity, comparative studies, and large scale investigations of plasma LCATase, as well as studies of the mechanism and regulation ofLCATase reaction.

299 citations


Journal ArticleDOI
TL;DR: A theoretical model is proposed to explain how the increase in mitochondrial protein concentration, and therefore of the oxidative enzymes, that occurs with endurance training could operate to alter the choice of substrate during submaximal exercise in a manner such that the oxidation of fatty acids increases, glycogen depletion and lactate production are reduced, and work capacity is enhanced.
Abstract: A theoretical model is proposed to explain how the increase in mitochondrial protein concentration, and therefore of the oxidative enzymes, that occurs with endurance training could operate to alter the choice of substrate during submaximal exercise in a manner such that the oxidation of fatty acids increases, glycogen depletion and lactate production are reduced, and work capacity is enhanced. The model is based on the control of enzyme activities both by enzyme and substrate concentrations. The effect of altering enzyme concentration on reaction velocities is presented on the basis of standard Henri-Michaelis-Menten kinetics. It is shown that the reaction velocity at a given substrate concentration is a function of total enzyme concentration. With an increase in total enzyme concentration there is a parallel increase in reaction velocity at the same substrate level. This would have its greatest impact at substrate levels below the Km of the enzyme. It would have an effect of enhancing fatty acid flux through the oxidative pathways while inhibiting the Embden-Meyerhof pathway. The model, as proposed, is consistent with known alterations in metabolism as they occur in man during submaximal exercise following endurance training.

278 citations


Book ChapterDOI
TL;DR: A further result is that the fate of oxygen in the catalysis can be followed and the rate of release from the enzyme of water bearing substrate oxygen, when the substrate is 18 O-labeled bicarbonate is revealed.
Abstract: Publisher Summary This chapter reviews the application of an 18 O exchange method to study the catalysis of the hydration of CO 2 by carbonic anhydrase, which, with a maximal turnover number near 10 6 sec –1 , is one of the fastest known enzymic reactions. The catalysis is so rapid that proton transfer steps between the enzyme and its environment, which are not ordinarily rate limiting for slower enzymic reactions, become rate-limiting and more amenable to investigation with carbonic anhydrase. The 18 O exchange method is chosen to investigate this problem because it is an equilibrium method that may be used in the absence of buffer or in the presence of excess buffer concentration, and buffers are sources of protons for transfer to the enzyme. With this method, it is possible to elucidate the role of proton transfer in the pathway of the catalytic hydration of CO 2 . A further result is that the fate of oxygen in the catalysis can be followed; hence, this method reveals the rate of release from the enzyme of water bearing substrate oxygen, when the substrate is 18 O-labeled bicarbonate.

203 citations


Journal ArticleDOI
TL;DR: It was concluded that most of the O2(-) originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide.
Abstract: The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of O2(-) radicals by microsomes, a stoichiometry of O2(-) to H2O2 close to 2:1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of H2O2. The rates of O2(-) as well as H2O2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%. It was concluded that most of the O2(-) originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 microM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.

199 citations


Journal ArticleDOI
TL;DR: The precise definition of this acetyl-transferase is of primary importance for the development of new pharmacological agents capable of moduling a potent platelet aggregating factor.

170 citations



Journal ArticleDOI
TL;DR: The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products and can be adequately described by a single equilibrium.

Journal ArticleDOI
TL;DR: Kinetic studies show a biphasic rate for Enzyme I phosphorylation, suggesting that the enzyme is phosphorylated in the associated state, and the phosphate transfer potentials of the PTS phosphoproteins are among the highest of known biological phosphate derivatives.

Patent
13 May 1982
TL;DR: In this article, a multisubstrate enzyme was incorporated into a thin layer electrochemical cell laminate having exterior membrane layers and an interior enzyme layer, and a control electrode located within the enzyme layer applied an electrical potential to the enzyme.
Abstract: A method for control of relative enzyme activity for analytical purposes. The activity of a multisubstrate enzyme may be controlled as a function of the electrical potential applied to the enzyme. The enzyme is preferrably incorporated into a thin layer electrochemical cell laminate having exterior membrane layers and an interior enzyme layer. A control electrode located within the enzyme layer applies an electrical potential to the enzyme. An intermediate electron transfer agent may be used to transfer electrons to and from the enzyme and the control electrode.

Journal ArticleDOI
TL;DR: Studies on the positional specificity of the enzyme showed that the preferred site of hydrolysis was sn-3 and sn-1, although a good percentage of the sn-2 position was also hydrolysed, and some of the properties of the purified human gastric juice acid lipase resembles those of rat and human lingual lipase.

Journal ArticleDOI
TL;DR: A low-molecular-weight human liver acid phosphatase was purified 2580-fold to homogenity by a procedure involving ammonium sulfate fractionation, acid treatment, and SP-Sephadex ion-exchange chromatography with ion-affinity elution.

Patent
15 Mar 1982
TL;DR: An immobilized enzyme electrode is used to measure the substrate concentration of an enzyme and in conversion from enzyme reaction energies into electric energies as discussed by the authors, which allows the determination quantity of the enzyme substrate as extremely low as approximately 10 -5 to 10 -6 mole/l in concentration.
Abstract: An immobilized enzyme electrode effective in measurement of the substrate concentration of the enzyme and in conversion from enzyme reaction energies into electric energies. The immobilized enzyme, of an oxidase system, such as glucose oxidase, amino acid oxidase, xanthine oxidase or the like and a metal oxide capable of constituting a redox system which is reduced through coupling with these enzyme reactions and is electrochemically oxidized (anodic oxidation) are combined with each other. The use of the enzyme electrode allows the determination quantity of the enzyme substrate as extremely low as approximately 10 -5 to 10 -6 mole/l in concentration.

Journal ArticleDOI
TL;DR: Urease of high specific activity is commercially available and because it does not occur in mammalian tissues, it is suitable for use in EIA tests to detect cell-associated antigens and their antibodies.

Journal ArticleDOI
TL;DR: A comparative study of angiotensin-converting enzyme activity in plasma, kidney and lung of five experimental animals showed a high degree of variation from species to species.

Journal ArticleDOI
TL;DR: Malic enzyme of pigeon liver catalyzes metal activated component reactions: Oxalacetate decarboxylase; reductase with broad specificity on α-ketocarboxylic acids; a NADP+-dependent dismutation of L-malate to L-lactate; and proton exchange between pyruvate and medium water.
Abstract: Malic enzyme of pigeon liver is a tetrameric molecule with identical, or nearly-identical subunits. It catalyzes, in addition to oxidative decarboxylation of L-malate, the following metal activated component reactions: Oxalacetate decarboxylase; reductase with broad specificity on α-ketocarboxylic acids; a NADP+-dependent dismutation of L-malate to L-lactate; and proton exchange between pyruvate and medium water. The kinetic mechanism of oxidative decarboxylase is sequential and ordered, with NADP+ adding first to the metal enzyme, followed by L-malate, and by the release of products CO2, pyruvate, and NADPH. NADPH release, or a conformation change preceeding it, is rate-limiting in the overall reaction. Chemical modification studies indicate the presence of histidyl and lysyl residues at the nucleotide site, and tyrosyl residues at the carboxylic acid site. The involvement of protonated histidine(s) in NADPH binding is implicated by results of direct titration experiments, which also suggest a role of this residue as a proton sink in the catalytic reaction. A cysteinyl SH group is located near (but not at) each of the substrate-sites on the enzyme tetramer. Reaction of these groups with SH reagents causes selective loss of activities involving decarboxylation (i.e., oxidative decarboxylase, reductive carboxylase, and oxalacetate decarboxylase), owing to blockage of the reversible carbon-carbon cleavage step by the bulky substituent. All-of-the-sites reactivity is observed for non-specific thiol reagents such as 5,5′ dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetate, and iodoacetamide. While bromopyruvate, which is reduced by the enzyme to L-bromolactate under catalytic conditions, alkylates these groups in an active-site directed manner with half-of-the-sites stoichiometry. The remaining two SH groups are reactive toward non-specific reagents, but at rates 2.4 - 3.6 fold lower than do the same groups on the unalkylated enzyme. This behavior is interpreted in terms of the ligand-induced negative cooperativity concept of Koshland, et al. (Biochemistry 5: 365–385, 1966): Reaction of bromopyruvate induces a conformation change on the alkylated subunit which is transmitted to the unoccupied subunit neighbor, turning off its catalytic site for reaction with L-malate, as well as converting the initial ‘fast’ SH groups into ‘slow’, or unreactive SH groups. In equilibrium binding experiments, all-of-the-sites reactivity is seen with nucleotide cofactors NADP+ and NADPH. Binding of Mn2+, or L-malate in the presence of Mn2+ and NADPH is biphasic, showing two ‘tight’ sites with dissociation constants in the micromolar range, and two ‘weak’ sites with 10–100 fold lower affinities. The presence of ‘tight’ and ‘weak’ L-malate sites is confirmed by fluorescence titration experiments which also yields similar affinities for the substrate molecule. In kinetic studies, two types of non-equivalent, and functionally distinct sites are detected. At saturating NADP+, and Mn2+ and L-malate levels corresponding to binding at tight sites, typical Michaelian behavior is observed. The reaction is inhibited uncompentitively by L-malate at higher concentrations corresponding to occupancy at all of the L-malate sites. Occupancy of Mn2+ at weak metal sites as well has no effect at low L-malate, but prevents substrate inhibition at high L-malate. A tentative ‘half-of-the-sites’ model consistent with results of chemical modification, binding, and kinetic experiments is proposed for this enzyme. This model implicates involvement of subunit cooperativity in the catalytic process. Malic enzyme is depicted as a tetramer composed of inititally identical subunits, each containing an active-site capable of binding all reactants. Mn2+ and L-malate bind anticooperatively to the tight and weak sites, in contrast to NADP+ which binds equivalently to all sites. On the fully active enzymes, only half (or the tight) of the subunits are simultaneously undergoing catalysis. Binding of L-malate (but not Mn2+) at the adjacent weak subunits causes a slow isomerization of the enzyme, and inhibition of NADPH dissociation from the catalytic subunits. Binding of Mn2+ at the same sites prevents this change and thereby relieving substrate inhibition. This model is further supported by results of active-site titration experiments, such as the half-size burst of enzyme-bound NADPH in the transient state, and half-of-the-sites reactivity of oxalate, an analog for the transition state intermediate of the reaction.

Journal ArticleDOI
TL;DR: 2,4-Dichlorophenol hydroxylase has been purified 13-fold from Acinetobacter grown on 2, 4-diclorophenoxyacetic acid as sole carbon source and possesses broad effector specificity.
Abstract: 1 2,4-Dichlorophenol hydroxylase has been purified 13-fold from Acinetobacter grown on 2,4-dichlorophenoxyacetic acid as sole carbon source. The enzyme was estimated to be 80–90% pure by electrophoresis. 2 The enzyme has a relative molecular mass of about 240000 and consists of four subunits of identical size. 3 The enzyme cont,ains FAD as the prosthetic group. FAD could not be replaced by riboflavin or FMN in reconstituting active enzyme from apoenzyme. 4 The reaction catalysed is an NADPH-dependent hydroxylation of 2,4-dichlorophenol with the formation of 3,5-dichlorocatechol as product. The reaction stoichiometry is typical of a monooxygenase with an external electron donor. NADPH is the preferred reduced pyridine nucleotide substrate but the enzyme can function with NADH. 5 The enzyme possesses broad effector specificity. In addition to 2,4-dichlorophenol, 4-chlorophenol and 4-chloro-2-methylphenol are true substrates for the enzyme. A number of ‘non-substrate effectors’ has been found. 6 The enzyme is sensitive to thiol-inhibiting reagents.

Journal ArticleDOI
TL;DR: The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta- GLUCosidases from other organisms being studied for use in industrial cellulose saccharification.
Abstract: A β-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50°C. The β-glucosidase had Km values of 0.78 and 0.40 mM for p-nitrophenyl-β-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a Ki of 3.5 mM when p-nitrophenyl-β-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-β-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The β-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated β-glucosidase exhibited for cellobiose compared favorably with the values obtained for β-glucosidases from other organisms being studied for use in industrial cellulose saccharification.

Journal ArticleDOI
TL;DR: One of the two species present in cyanide solutions, CN- was shown to be a potent reversible inhibitor of total electron flow, apparently uncoupling MgATP hydrolysis and electron transfer, and extrapolation indicates that at high enough HCN concentration H2 evolution can be eliminated.
Abstract: We have examined the reduction of cyanide by using the purified component proteins of nitrogenase (Av1 and Av2). The previously reported self-inhibition phenomenon was found to be an artifact. One of the two species present in cyanide solutions, CN-, was shown to be a potent reversible inhibitor (Ki = 27 microM) of total electron flow, apparently uncoupling MgATP hydrolysis and electron transfer. There appears to be no differential effect of CN- on the specific activities of Av1 and Av2 nor is there any apparent irreversible physical damage to Av2. CN- inhibition is completely reversed by low levels of CO, implying a common binding site. Azide partially relieves the inhibitory effect, but other substrates and inhibitors (N2, C2H2, N2O, H2) have no effect. The other species present in cyanide solutions, HCN, was shown to be the substrate (Km = 4.5 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough HCN concentration H2 evolution can be eliminated. The products are methane plus ammonia (six electrons), and methylamine (four electrons). There is an excess (relative to methane) of ammonia formed, which, according to electron balance studies, may arise from a two-electron intermediate. Both nitrous oxide and acetylene (but not N2) influence the distribution of cyanide reduction products, implying simultaneous binding. HCN appears to bind to and be reduced at an enzyme state more oxidized than the one responsible for either H2 evolution or N2 reduction.

Journal ArticleDOI
TL;DR: A high-resolution x-ray crystallographic investigation of the complex between carboxypeptidase A and the slowly hydrolyzed substrate glycyl-L-tyrosine confirmed that this substrate may be bound in a nonproductive manner, because the hydrolytically important zinc-bound water has been displaced and excluded from the active site.
Abstract: A high-resolution x-ray crystallographic investigation of the complex between carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the slowly hydrolyzed substrate glycyl-L-tyrosine was done at -9 degrees C. Although this enzyme-substrate complex has been the subject of earlier crystallographic investigation, a higher resolution electron-density map of the complex with greater occupancy of the substrate was desired. All crystal chemistry (i.e., crystal soaking and x-ray data collection) was performed on a diffractometer-mounted flow cell, in which the crystal was immobilized. The x-ray data to 1.6-A resolution have yielded a well-resolved structure in which the zinc ion of the active site is five-coordinate: three enzyme residues (glutamate-72, histidine-69, and histidine-196) and the carbonyl oxygen and amino terminus of glycyl-L-tyrosine complete the coordination polyhedron of the metal. These results confirm that this substrate may be bound in a nonproductive manner, because the hydrolytically important zinc-bound water has been displaced and excluded from the active site. It is likely that all dipeptide substrates of carboxypeptidase A that carry an unprotected amino terminus are poor substrates because of such favorable bidentate coordination to the metal ion of the active site.

Journal ArticleDOI
TL;DR: A model is proposed in which Cl- has a dual role, acting both as a substrate and as an inhibitor, and the rate equation derived from this model is used to derive values of Km for H2O2 in good agreement with the experimentally determined values.

Book ChapterDOI
TL;DR: This chapter describes the assay method, purification, and properties of alcohol dehydrogenase isolated from acetic acid bacteria, which differs from alcohol dehydrogensase of methanol utilizingacteria, which without exception requires ammonia for full activity.
Abstract: Publisher Summary This chapter describes the assay method, purification, and properties of alcohol dehydrogenase isolated from acetic acid bacteria. Alcohol dehydrogenase of acetic acid bacteria acts on a wide range of primary alcohols, except methanol. The enzyme acts as a vinegar producer by coupling with aldehyde dehydrogenase. The enzyme is localized on the outer surface of the cytoplasmic membrane and the oxidation of substrate is linked to its respiratory chain. The enzyme differs from alcohol dehydrogenase of methanol utilizing bacteria, which without exception requires ammonia for full activity. The reaction rate is estimated (a) by spectrophotometry in the presence of 2,6-dichlorophenolindophenol and phenazine methosulfate; (b) by colorimetry in the presence of potassium ferricyanide; (c) by polarography with an oxygen electrode; or (d) by manometry in a conventional Warburg apparatus. The assay method with potassium ferricyanide is employed because of its simplicity for routine assay. The steps involved in the purification of alcohol dehydrogenase from G. suboxydans and A. aceti are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) diethylaminoethyl (DEAE)-Sephadex column chromatography I and II, and (4) hydroxyapatite column chromatography.

Journal ArticleDOI
TL;DR: 2-Phosphinomethylmalic acid synthase catalyzes the condensation of phosphinopyruvic acid, an analog of oxalacetic acid, and acetyl-CoA to form PMM, which is very similar to (R)-citrate synthase of Clostridium in the inhibition pattern by sulfhydryl compounds, its metal ion requirement and stereospecificity.
Abstract: 2-Phosphinomethylmalic acid (PMM) synthase catalyzes the condensation of phosphinopyruvic acid (PPA), an analog of oxalacetic acid, and acetyl-CoA to form PMM. The enzyme was purified approximately 700-fold from a cell-free extract of Streptomyces hygroscopicus SF1293, a bialaphos producing organism, to an electrophoretically homogeneous state. The purified PMM synthase has a subunit molecular weight of 48, 000 by SDS-polyacrylamide gel electrophoresis and a native molecular weight of 90, 000 - 98, 000 by gel filtration. PMM synthase was relatively unstable, showed maximum activity at pH 8.0 and 30°C, and was inhibited strongly by p-chloromercuribenzoate, iodoacetamide and EDTA. Enzyme activity suppressed by EDTA was completely restored by adding Co+ + or Mn+ + and partially restored by addition of Ca+ +, Fe+ + or Mg+ +. The specific substrates of this enzyme are PPA or oxalacetic acid in addition to acetyl-CoA. The enzyme does not catalyze the liberation of Co A from acetyl-CoA in the presence of α-keto acids, such as pyruvate, α-ketoglutarate, deamino-α-ketodemethylphosphinothricin or phosphonopyruvate. The condensation reaction did not take place when propionyl-CoA or butyryl-CoA was used as a substrate in place of acetyl-CoA. The Km values of the enzyme were 0.05 mM for acetyl-CoA, 0.39 mM for PPA and 0.13 mM for oxalacetate. PMM synthase is very similar to (R)-citrate synthase of Clostridium in the inhibition pattern by sulfhydryl compounds, its metal ion requirement and stereospecificity; unlike CR)-citrate synthase PMM synthase was not inhibited by oxygen.

Journal ArticleDOI
TL;DR: Kinetic and spectral data establish that peroxidase may oxidize indole-3-acetic acid by either of two pathways depending on the enzyme/substrate ratio, and important biological consequences may follow activation of this shuttle under physiological conditions.
Abstract: Kinetic and spectral data establish that peroxidase may oxidize indole-3-acetic acid by either of two pathways depending on the enzyme/substrate ratio. When relatively low enzyme/substrate ratios are employed, the oxidation proceeds through a reduced peroxidase in equilibrium compound III shuttle. Conversely, peroxidase operates through the conventionally accepted pathway involving native enzyme and compounds I and II only when high enzyme/substrate ratios are used. Compound III, a specific oxidase, constitutes the dominant steady-state form of peroxidase when the reduced peroxidase in equilibrium compound III shuttle is operational. Activation of this shuttle also produces a flux of superoxide anion radical at the expense of molecular oxygen. Thus, important biological consequences may follow activation of this shuttle under physiological conditions.

Journal ArticleDOI
TL;DR: The CPA-catalyzed hydrolysis of Z-Glys-Phe was shown to involve only C-N bond cleavage, to give carbobenzoxythioglycine and phenylalanine.
Abstract: Carbobenzoxythioglycyl-L-phenylalanine [CbzNHCH2C(==S)Phe, Z-Glys-Phe] was synthesized as thioamide analogue of Z-Gly-Phe, a known substrate of carboxypeptidase A (CPA). By use of a ninhydrin-based assay and Z-Gly-Gly-Phe as the substrate, Z-Glys-Phe was shown to be a weak competitive inhibitor of CPA (Ki = 1.4 mM). The L isomer (but not the D) of Z-Glys-Phe proved to be a substrate for CPA (Km = 1.1 mM and kcat = 5.3 s-1 at pH 7.5), binding with comparable affinity to, but hydrolyzing at 10% the rate of, the oxo analogue Z-Gly-Phe. The CPA-catalyzed hydrolysis of Z-Glys-Phe was shown to involve only C-N bond cleavage, to give carbobenzoxythioglycine and phenylalanine.

Journal ArticleDOI
TL;DR: It is found that the novel substrates, methionine and norleucine, are also able to activate the enzyme, and the extent of the change in substrate specificity on activation of the enzyme is much greater than had been heretofore realized.