Isatin inhibition of rat testicular alkaline phosphatase. A non-allosteric phenomenon.

Isatin has been found to inhibit rat testicular alkaline phosphatase (EC 3.1.3.1) uncompetitively. The hyperbolic curve relating inhibition as a function of substrate concentration; the persistence of inhibition after the tertiary structure of the enzyme has been altered by heat denaturation, exposure to urea or papain digestion; the small changes in entropy, free energy and enthalpy in the presence of isatin, and the number of isatin molecules (n = 1.29) combining with one molecule of enzyme indicate the non-allosteric nature of inhibition.     

Isatin-enzyme interactions. VI. Inhibition of rat kidney alkaline phosphatase.]

Interaction of isatin with rat kidney alkaline phosphatase has been studied. Mode of attachment of isatin with the enzyme protein is most likely through amino group(s), which is also imperative for catalysis. Sulphydryl group(s) do not seem to be involved in enzyme action. Zinc is also needed for enzyme activity. Use of sulphydryl compounds suggests that isatin inhibition of the enzyme is through attachment at the metal site. However, this inhibition may not only be due to simple chelation of the metal by isatin.     

Glycosides of hydroxylamine derivatives. I. phase transfer synthesis and study of isatine-3-oximes glucosaminides influence on bacterial luminescence

In the phase transfer system solid calcium carbonate-acetonitrile, per acetate alpha-D-glucosaminilchloride glycosilate easily deprotoned isatine-3-oximes hydroxyl groups. It was found that the presence in the reaction mixture a catalytic amounts of 15-crown-5 accelerated the process twice. Obtained O-beta-D-glucosaminides were identified with 1H-NMR spectroscopy. Features of synthesized compound's NMR spectra are discussed in comparison with those of another N-acetylglucosamine 1-O-derivatives. The biological activity of the some oximes with different substituents in isatin residuum has been studied in a test of inhibition of bioluminescence of marine luminescent bacteria Photobacterium leiognathi Sh12. The nature of N-substituent of isatin fragment and 5-substituent of isatin main structure is compared with glycosides ability to suppress bacterial luminescence.     

Interaction of pyruvate kinase with isatin and deprenyl

The key glycolytic enzyme, pyruvate kinase, exhibits moderate affinity [³H]isatin binding (K_D ～10 μM) which is inhibited by ATP (IC₅₀ 25 μM) and deprenyl (IC₅₀ 5 μM). Interaction of pyruvate kinase with isatin and its inhibition by ATP and deprenyl has also been confirmed using an independent biosensor technique and the immobilized isatin analogue, aminoisatin. This effect has some specificity because the enzyme, creatine phosphokinase, does not exhibit specific isatin-binding. It is suggested that interaction of pyruvate kinase with isatin may reflect some non-glycolytic functions of this enzyme.     

Characterization of a major form of human isatin reductase and the reduced metabolite.

Isatin, an endogenous indole, has been shown to inhibit monoamine oxidase, and exhibit various pharmacological actions. However, the metabolism of isatin in humans remains unknown. We have found high isatin reductase activity in the 105,000 g supernatants of human liver and kidney homogenates, and have purified and characterized a major form of the enzyme in the two tissues. The hepatic and renal enzymes showed the same properties, including an M(r) of 31 kDa, substrate specificity for carbonyl compounds and inhibitor sensitivity, which were also identical to those of recombinant human carbonyl reductase. The identity of the isatin reductase with carbonyl reductase was immunologically demonstrated with an antibody against the recombinant carbonyl reductase. About 90% of the soluble isatin reductase activity in the liver and kidney was immunoprecipitated by the antibody. The Km (10 microm) and k(cat)/K(m) (1.7 s(-1) x microm(-1)) values for isatin at pH 7.0 were comparable to those for phenanthrenequinone, the best xenobiotic substrate of carbonyl reductase. The reduced product of isatin was chemically identified with 3-hydroxy-2-oxoindole, which is also excreted in human urine. The inhibitory potency of the reduced product for monoamine oxidase A and B was significantly lower than that of isatin. The results indicate that the novel metabolic pathway of isatin in humans is mediated mainly by carbonyl reductase, which may play a critical role in controlling the biological activity of isatin.     

Inhibition of NO-dependent soluble human platelet guanylate cyclase by isatin

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. Physiologically relevant concentrations of isatin (ranged from 1 nM to 10 μM) did not influence basal activity of soluble human platelet guanylate cyclase (sGC), but caused a bell-shaped inhibition of the NO-activated enzyme. Inhibition of the NO-dependent activation by isatin did not depend on a chemical nature of the NO donors. The inhibitory effects of ODC (a heme-dependent inhibitor of sGC) and isatin were non-additive suggesting that the inhibitory effect of isatin may involve the heme binding domain (possibly heme iron) and experiments with hemin revealed some isatin-dependent changes in its spectrum. Isatin also inhibited sGC activation by the allosteric activator YC-1. It is suggested that the bell shaped inhibition of the NO-dependent activation of sGC by isatin may be attributed to complex interaction of isatin with the heme binding domain and the allosteric YC-1-binding site of sGC.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times

The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, emphasizing the kinetic characteristics associated with the extended hydrolysis times. The changes in the hydrolysis rate and extent of soluble protein adsorption during the progress of reaction, either apparent or intrinsic, were investigated. The hydrolysis rate declined drastically during the initial hours of hydrolysis. The factors causing the reduction in the hydrolysis rate were examined; these include the transformation of cellulose into a less digestible form and product inhibition. The structural transformation can be partially explained by changes in the crystallinity index and surface area. The product inhibition was caused by the deactivation of the adsorbed soluble protein by the products, which essentially represents the so-called "un-competitive" inhibition. The kinetics of beta-glucosidase were also studied. The result has shown that the action of beta-glucosidase is competitively inhibited by glucose. It has been found that the integrated form of the initial rate expression cannot be used in predicting the progress of reaction because the digestibility of cellulose changes drastically as the hydrolysis proceeds, and that the rate expression for enzymatic hydrolysis of cellulose cannot be simplified or approximated by resorting to the pseudo-steady-state assumption. A mechanistic kinetic model of cellulose hydrolysis should include the following major influencing factors: (1)mode of action of enzyme, (2) structure of cellulose, and (3) mode of interaction between the enzyme and cellulose molecules.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

Microbial transformation of (-)-isolongifolol and butyrylcholinesterase inhibitory activity of transformed products

The microbial transformation of (-)-isolongifolol (1) by using the standard two-stage fermentation technique with Fusarium lini afforded polar oxygenated metabolites: 10-oxoisolongifolol (2), 10alpha-hydroxyisolongifolol (3), and 9alpha-hydroxyisolongifolol (4). Metabolites 3 and 4 were also formed with the incubation of 1 with Aspergillus niger. All three metabolites were found to be new. Compounds 3 and 4 inhibited butyrylcholinesterase enzyme in a concentration-dependent manner with IC50 values 13.6 and 299.5 microM, respectively. Compound 3 showed un-competitive mode of inhibition against butyrylcholinesterase with Ki value 15.0 microM. The structures of metabolites 2-4 were deduced on the basis of spectroscopic techniques and single-crystal X-ray diffraction techniques.     

Alkaline phosphatase from Echinococcus multilocularis: purification and characterization.

1. Kinetic and physical parameters of purified alkaline phosphatase from Echinococcus multilocularis metacestodes, livers of infected gerbils and control animals were determined. 2. Km value for p-nitrophenyl phosphate was about 0.05 +/- 0.02 mM for the three enzymes. 3. Vmax values were 357 +/- 67 nmol/min/mg proteins for metacestode enzyme, and 6.7 +/- 1.1 and 6.7 +/- 0.8 nmol/min/mg proteins for liver enzyme of infected and control animals, respectively. 4. Mr and pI were different for the parasite and hepatic enzyme. 5. The parasite enzyme was less sensitive to the elevation of temperature than hepatic enzyme. 6. The isatin inhibition was a competitive inhibition type for parasite and uncompetitive type for host liver enzyme.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by 5-nitroisatin and the antiviral agent arbidol

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. The effect of isatin derivatives, 5-nitroisatin and arbidol (an antiviral agent) on spermine NONO-induced activation of human platelet soluble guanylate cyclase has been investigated. 5-Nitroisatin and arbidol had no effect on basal activity, but synergistically increased in a concentration-dependent manner the spermine NONO-induced activation of this enzyme. 5-Nitroisatin and arbidol, like YC-1, sensitized guanylate cyclase towards nitric oxide (NO) and produced a leftward shift of the spermine NONO concentration response curve. However, both compounds did not influence the activation of guanylate cyclase by YC-1 and did not change the synergistic increase of spermine NONO-induced activation of soluble guanylate cyclase in the presence of YC-1. This suggests that 5-nitroisanin and arbidol did not compete with YC-1. Addition of isatin did not change the synergistic increase in the spermine NONO-induced guanylate cyclase activation by 5-nitroisatin and arbidol and did not influence a leftward shift of the spermine NONO concentration response curve produced by these compounds. These data suggest lack of competitive interaction between isatin and both its derivatives used.     

Ketopantoyl lactone reductase is a conjugated polyketone reductase

Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.     

Isatin causes a rapid accumulation of ATP in synaptosomes: Implication for stress and regulation of natriuretic peptide receptors

Isatin is an endogenous indole, which is increased in mammalian brain and peripheral tissues under conditions of stress. Physiological concentrations of isatin inhibit natriuretic peptide (NPR) receptor binding and NPR-dependent signalling. The inhibition of NPR signalling by isatin is attenuated by a nonhydrolyzable ATP analogue. In this study we have demonstrated that short term incubation of rat brain synaptosomes with a physiological concentration of isatin caused a rapid 3-fold accumulation of ATP. The additional increase of ATP in the presence of tyrphostin, an inhibitor of tyrosine kinase, which uses ATP for phosphorylation of some proteins, suggests the dependence of this phenomenon on the activity of ATP-consuming systems. ATP inhibited binding of [³H]isatin to both particulate and soluble fractions of the rat brain. These results suggest that isatin induces accumulation of ATP, which in turn may displace isatin from both membrane-bound and soluble binding sites. Since natriuretic peptides are known to decrease stress hormone release this regulatory loop may be involved in the maintenance of natriuretic peptide signalling under conditions of stress and thus contribute to the control of stress responses.     

Metabolism of aromatic compounds by fungi. Kinetic properties and mechanism of 3-carboxy-cis,cis-muconate cyclase from Aspergillus niger.

A preliminary investigation of the kinetic properties of 3-carboxy-cis,cis-muconate cyclase (EC 5.5.1.5) has been performed. The initial velocity of the reaction was shown to be proportional to the concentration of the enzyme in the assay system adopted and the apparent Km was found to be 57 muM at pH 6.0 and 30 degrees C but at concentrations exceeding 70 muM, substrate inhibition was apparent. At pH 6.0 the Ki for the substrate was 0.45 mM. Plots of V and Km against pH showed inflexions at pH 5.3 and pH 6.4. The enzyme was inhibited by a variety of inorganic anions and by a number of dicarboxylic and tricarboxylic acids. The degree of inhibition exerted by these acids was found to be proportional to the proximity of their carboxyl groups, the cis configuration being a more effective inhibitor than the trans configuration. As inhibition was competitive in each case, the presence of an anion-sensitive substrate-binding site has been postulated. The cis-cis, cis-trans and trans-trans isomers of muconate, 3-chloromuconate and 3-carboxy-cis-trans-muconate, close analogues of natural substrate but not attacked by the enzyme, were also found to be competitive inhibitors. The variation in pKi with pH was determined in the case of cis,cis-muconate and cis-aconitate, both of which gave curves suggesting the importance of a group with a pKa of approximately 6.4 responsible for increasing the inhibition of the enzyme. Modification by ethoxyformic anhydride and the kinetics of Rose-Bengal-sensitized photo-oxidation suggested the participation of a histidine residue in the catalytic reaction. These results are discussed in the light of recent work on enzymes catalysing analogous reactions; a likely reaction mechanism has been proposed.     

Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase

Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.     

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7? distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.     

The nature of the differential inhibition of polyphenylalanine synthesis in extracts from Artemia salina and rabbit reticulocytes by the Phytolacca americana protein

The difference in sensitivity of polyphenylalanine synthesis in extracts from Artemia salina and rabbit reticulocytes to inhibition by the Phytolacca americana protein (PAP) has been found to be linked to the source of the supernatant enzyme fraction and not the ribosomes. In the presence of reticulocyte supernatant enzyme fraction polyphenylalanine synthesis is less sensitive to inhibition by PAP than that observed in the presence of A. salina supernatant enzyme fraction. The results suggest that reticulocyte elongation factor 2 is responsible for this effect.     

A novel fungal enzyme, NADPH-dependent carbonyl reductase, showing high specificity to conjugated polyketones. Purification and characterization

A novel enzyme which specifically catalyzes the reduction of conjugated polyketones was purified to homogeneity from cells of Mucor ambiguus AKU 3006. The enzyme has a strict requirement for NADPH and irreversibly reduces a number of quinones such as p-benzoquinone, alpha-naphthoquinone and acenaphthenequione. The enzyme also reduces polyketones such as isatin and ketopantoyl lactone, and their derivatives. The apparent Km values for isatin and ketopantoyl lactone are 49.9 microM and 714 microM, respectively. The reduction of ketopantoyl lactone proceeds stereospecifically to yield L-(+)-pantoyl lactone. The pro-S (A) hydrogen at C-4 of NADPH is transferred to the substrate. The enzyme is not a flavoprotein and consists of two polypeptide chains with an identical relative molecular mass of 27,500. Quercetin, dicoumarol and some SH reagents inhibit the enzyme activity. 3-Methyl-1,2-cyclopentanedione and 1,3-cyclohexanedione are uncompetitive inhibitors with Ki values of 80.9 microM and 64.5 microM, respectively, to ketopantoyl lactone.     

The self-association of jack bean urease and its modification by silver ions.

At low ionic strength urease has been found to dissociate at protein concentrations below 1 × 10⁸m. The inhibition of enzyme activity by Ag⁺ has been used to demonstrate this. The inhibition by Ag⁺ has been shown to be independent of dissociation but, at dilutions where dissociation occurs, silver ion modifies the process. Urease is aggregated by Ag⁺ at high Ag⁺:protein ratios. Such inactive aggregates can be solubilized and reactivated by dithiothreitol. Further evidence has been obtained indicating the similarity of the (8n) and (16n) forms of urease. The phenomena of inhibition and aggregation in the presence of the heavy metal ion have been shown to be separate processes.     

Selective enhancement of the activity of C-terminally truncated, but not intact, acetylcholinesterase

Acetylcholinesterase (AChE) is one of the fastest enzymes approaching the catalytic limit of enzyme activity. The enzyme is involved in the terminal breakdown of the neurotransmitter acetylcholine, but non-enzymatic roles have also been described for the entire AChE molecule and its isolated C-terminal sequences. These non-cholinergic functions have been attributed to both the developmental and degenerative situation: the major form of AChE present in these conditions is monomeric. Moreover, AChE has been shown to lose its typical characteristic of substrate inhibition in both development and degeneration. This study characterizes a form of AChE truncated after amino acid 548 (T548-AChE), whose truncation site is homologue to that of a physiological form of T-AChE detected in fetal bovine serum that has lost its C-terminal moiety supposedly due to proteolytic cleavage. Peptide sequences covered by this C-terminal sequence have been shown to be crucially involved in both developmental and degenerative mechanisms in vitro. Numerous studies have addressed the structure-function relationship of the AChE C-terminus with T548-AChE representing one of the most frequently studied forms of truncated AChE. In this study, we provide new insight into the understanding of the functional characteristics that T548-AChE acquires in solution: T548-AChE is incubated with agents of varying net charge and molecular weight. Together with kinetic studies and an analysis of different molecular forms and aggregation states of T548-AChE, we show that the enzymatic activity of T548-AChE, an enzyme verging at its catalytic limit is, nonetheless, apparently enhanced by up to 800%. We demonstrate, first, how the activity of T548-AChE can be enhanced through agents that contain highly positive charged moieties. Moreover, the un-competitive mechanism of activity enhancement most likely involves the peripheral anionic site of AChE that is reflected in delayed substrate inhibition being observed for activity enhanced T548-AChE. The data provides evidence towards a mechanistic and functional link between the form of AChE unique to both development and degeneration and a C-terminal peptide of T-AChE acting under those conditions.