Grundlagen der Bestimmung von Enzymaktivit?ten

Zusammenfassung Die optische Methode von Otto Warburg sowie andere physikalische Meßverfahren wie die Fluorimetrie, die Polarimetrie, die Titrimetrie bei stationäremph (ph-Stat) und andere Methoden werden heute bevorzugt zur Aktivitätsbestimmung von Enzymen herangezogen. Diese Verfahren, durch Anschluß registrierender Schreiber erleichtert, haben gegenüber den klassischen chemisch-analytischen Verfahren den Vorteil einer fortlaufenden Darstellung des Reaktionsablaufes und gestatten eine einfache Ablesung der Aktivität eines Enzyms als Maß seiner katalytischen Wirksamkeit.Die Anwendung direkter Meßverfahren wurde von Warburg erweitert durch die Darstellung und Verwendung hochgereinigter, kristallisierter Enzyme, die in zusammengesetzten Tests zur chemischen Kopplung eines Enzyms unbekannter Aktivität an ein sogenanntes Indicator-Enzym eingesetzt werden, dessen Aktivität direkt gemessen werden kann. Durch Einschaltung eines weiteren Hilfsenzyms kann schließlich die analytische Enzymkette verdreifacht werden.Grundbedingung der Aktivitätsbestimmung ist die Proportionalität von Reaktionsgeschwindigkeit und Enzymkonzentration. Sie wird nach der Theorie von Michaelis und Menten entweder bei Substratüberschuß oder bei sehr kleinen Substratkonzentrationen, oder empirisch, unabhängig von dem Ordnungstyp der Reaktion, durch Zeitmessung eines festgelegten Substratumsatzes gefunden. Die enzymatische Aktivität wird für definierte Standardbedingungen in internationalen Einheiten ausgedrückt. Die internationale Einheit ist als die Enzym-Menge definiert, die die Umwandlung von 1 Mol Substrat oder 1 Äquivalent einer reagierenden Gruppe oder gespaltenen Bindung bzw. das Auftreten von 1 Mol Produkt resp. Äquivalent unter Standardbedingungen je Minute katalysiert.

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Summary The optical method of Otto Warburg as well as other physical methods like fluorimetry, polarimetry,ph-stat-titrimetry among others, are nowadays preferred for activity determination of enzymes. These procedures which can be facilitated by direct recording have the advantage of a continuous registration of a course of a reaction and a direct determination of enzyme activities.The application of such direct physical methods has been extended by Warburg by the preparation and application of pure enzymes, which can be used in coupled tests for a chemical coupling of an enzyme of unknown activity with a so called indicator enzyme, the activity of which can directly be measured. By use of another accessory enzyme, a three-fold analytical enzyme sequence can be developed.The theoretical basis for enzyme determination is the proportionality of reaction rate and enzyme concentration. According to the theory of Michaelis and Menten, proportionality can be found either with optimal substrate concentration, or with very small substrate levels, and furthermore, empirically by time measurement of a fixed substrate utilization. — The activity of enzymes is expressed for standard conditions in international units. An international unit is the amount of enzyme which catalyzes the reaction of 1 Mole substrate or 1 Equivalent of a reacting group or split bondage, resp. the production of 1Mole product per minute.

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Wir danken der Deutschen Forschungsgemeinschaft, Bad Godesberg bei Bonn, für die Unterstützung unserer Arbeiten sowie Herrn Prof. E. Bartholomé für die Durchsicht des Manuskriptes.

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Vorgetragen auf der Tagung der Fachgruppe Analytische Chemie in Heidelberg im September 1963.     

Zur Kenntnis dermyo-Inosit-Oxygenase aus höheren Pflanzen

Zusammenfassung Wie bereits berichtet wurde¹, findet sich in Haferkeimlingen (Avena sativa) und in unreifen Erdbeerfrüchten (Fragaria) ein Enzym, dasmyo-Inosit, in gleicher Weise wie früher beschriebene Enzyme aus Rattennieren² und aus der HefeSchwanniomyces occidentalis ³ zud-Glucuronsäure abbaut. Eine nähere Untersuchung des Enzyms aus Haferkeimlingen ergab allerdings gewisse Unterschiede gegenüber den gleichspezifischen Enzymen anderer Herkunft. Die Affinität des Haferenzyms gegenübermyo-Inosit ist um eine Zehnerpotenz höher als diejenige der anderen Enzyme, das Enzym aus Hafer ist weniger empfindlich gegenüber manchen Inhibitoren und es zeigt eine viel breitere Spezifität. Es greift sämtliche in der Natur vorkommenden isomeren Inosite, eine Anzahl von Inositmono- und-dimethyläthern sowie verschiedene isomere Pentahydroxycyclohexanone an, wobei in allen Fällen Uronsäuren entstehen.

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It has been reported previously¹ that oats seedlings (Avena sativa) and immature strawberries (Fragaria) contain an enzyme catalyzing the degradation ofmyo-inositol tod-glucuronic acid in the same manner as known enzymes from rat kidney² and the yeastSchwanniomyces occidentalis ³. A closer investigation of the enzyme from oats showed, however, that it is somewhat different from the enzymes of other origin catalyzing the same reaction. The affinity of the oats enzyme towardsmyo-inositol is about one order of magnitude higher than that of the other enzymes, it is less sensitive to inhibitors, and it shows a broader specificity, attacking all naturally occurring inositol isomers, several mono- and dimethyl ethers of inositols, and several pentahydroxy-cyclchexanone isomers. All these compounds are degraded to uronic acids.

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Herrn Professor Dr.J. W. Breitenbach zu seinem 60. Geburtstag in freundschaftlicher Verehrung gewidemet.     

Poly(acrylic acid) is a common noncompetitive inhibitor for cationic enzymes with high affinity and reversibility

We have recently developed a versatile technique, complementary polymer pair system (CPPS), which enables switching the activity of diverse enzymes using anionic poly (acrylic acid) (PAAc) and cationic poly(allylamine) (PAA). To obtain a deeper understanding of CPPS, we investigated the manner by which PAAc inhibits cationic ribonuclease A, lysozyme, and trypsin. Studies of the enzyme kinetics showed that PAAc acts as a noncompetitive inhibitor for all these enzymes, and carries several potent enzyme binding sites (K_i ≈ 10^?8 M). In addition, the inhibited enzymes were recovered by oppositely charged PAA. These data indicate the generality of CPPS, as only the surface charge and not the substrate binding site of the enzymes should be considered when determining a charged polymer as an inhibitor. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Covalent attachment of trypsin on plasma polymerized allylamine

This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV–vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.     

Purification and Characterization of a Novel Heparin Degrading Enzyme from <Emphasis Type="Italic">Aspergillus flavus</Emphasis> (MTCC-8654)

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/μg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 °C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co²⁺, Mn²⁺ ions, and reducing agents (β-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K _m and V _max as 2.2 × 10⁻⁵ M and 30.8 mM min⁻¹, respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.     

Ein Beitrag zur viskosimetrischen Verfolgung der Kinetik bestimmter Enzymreaktionen und Bestimmung der Enzymaktivität

Zusammenfassung Es wurde eine Theorie aufgestellt, die es gestattet, die Kinetik bestimmter enzymatischer Hydrolysen und die Aktivität des daran beteiligten Enzyms zu verfolgen. Die spezif. Aktivitätk des Enzyms ist durch diejenige Reaktionsgeschwindigkeit gegeben, die durch die Einheit der Enzymmasse in der Volumseinheit pro Zeiteinheit bewirkt wird. Nach der hier aufgestellten Theorie hängt die spezif. Aktivität des Enzyms nicht vom Molekulargewicht und der Konzentration des verwendeten Substrates ab.

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A theory has been set for tracing the kinetics of some hydrolysis enzymes, which allows to determine the activity of the enzyme participating in them. The specific activityk of the latter is determined through the reaction rate in a unit of volume by a unit of enzyme mass for a unit of time (cm³·g^–1·sec^–1). According to this theory, the specific activity of the enzyme does not depend on the molecular weight and the concentration of the substrate used.

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Herrn Prof. Dr.T. Trandafilov zum 60. Geburtstag gewidmet.     

Glycosidases of turnip leaf tissues

Four myrosinase (β-thioglucosidase EC. 3.2.3.1) and seven disaccharase (β-fructofuranosidase, EC. 3.2.1.26) isoenzymes were isolated from turnip leaves. The most active enzymes were isolated in pure form. Myrosinase and disaccharase mol wt was 62.0 × 10³ and 69.5 × 10³ dalton, respectively, on the basis of gel filtration on Sephadex G-200. Myrosinase pH profile showed high activity between pH 5 and 7 with the optimum at pH 5.5. The purified enzyme was heat-stable for 60 min at 30°C with only loss of 24% of activity. Its activity is strongly inhibited (100%) by Pb²⁺, Ba²⁺, Cu²⁺ and Ca²⁺ ions, and activated (70%) by EDTA at 0.04M. The pure enzyme failed to hydrolyze amylose, glycogen, lactose, maltose, and sucrose. TheK _m andV _max values of myrosinase using sinigrin as specific substrate was 0.045 mM and 2.5 U, respectively. The maximal activity of disaccharase enzyme was obtained at pH 4–5 and 35–37°C. The enzyme was heat-stable at 30°C for 30 min with only 10% loss of its activity. Its activity is strongly activated (70–240%) by Ca²⁺, Ba²⁺, Cu²⁺, and EDTA at 0.01M. The enzyme activity is specific to the disaccharide sucrose and failed to hydrolyze other disaccharides (maltose and lactose). TheK _m andV _max of disaccharase were 0.123 mM and 3.33 U, respectively.     

Isolation and Characterization of a Novel Thermophilic-Organic Solvent Stable Lipase From <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis>

The benzene tolerant Acinetobacter baylyi isolated from marine sludge in Angsila, Thailand could constitutively secrete lipolytic enzymes. The enzyme was successfully purified 21.89-fold to homogeneity by ammonium sulfate precipitation and gel-permeable column chromatography with a relative molecular mass as 30 kDa. The enzyme expressed maximum activity at 60°C and pH 8.0 with p-nitrophenyl palmitate as a substrate and found to be stable in pH and temperature ranging from 6.0-9.0 to 60-80°C, respectively. A study on solvent stability revealed that the enzyme was highly resisted to many organic solvents especially benzene and isoamyl alcohol, but 40% inhibited by decane, hexane, acetonitrile, and short-chain alcohols. Lipase activity was completely inhibited in the presence of Fe²⁺, Mn²⁺, EDTA, SDS, and Triton X-100 while it was suffered detrimentally by Tween 80. The activity was enhanced by phenylmethylsulfonyl fluoride (PMSF), Na⁺, and Mg²⁺ and no significant effect was found in the presence of Ca²⁺ and Li⁺. Half of an activity was retained by Ba²⁺, Ag⁺, Hg⁺, Ni²⁺, Zn²⁺, and DTT. The enzyme could hydrolyze a wide range of p-nitrophenyl esters, but preferentially medium length acyl chains (C₈-C₁₂). Among natural oils and fats, the enzyme 11-folds favorably catalyzed the hydrolysis of rice bran oil, corn oil, sesame oil, and coconut oil in comparison to palm oil. Moreover, the transesterification activity of palm oil to fatty acid methyl esters (FAMEs) revealed 31.64 ± 1.58% after 48 h. The characteristics of novel A. baylyi lipase, as high temperature stability, organic solvent tolerance, and transesterification capacity from palm oil to FAMEs, indicate that it could be a vigorous biocatalyzer in the prospective fields as bioenergy industry or even in organic synthesis and pharmaceutical industry.     

Bienzyme Electrode Compatible Behavior for Water-Soluble and-Insoluble Substrates

Abstract

A bienzymatic sensing layer containing two enzymes able to work sequentially, choline oxidase (ChOD) and phospholipase D (PLaseD), was used to design an electrochemical biosensor for the detection of either a water-soluble (choline) or insoluble (phosphatidylcholine) substrate. A photocrosslinkable polymer, poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), was used as host-matrix for enzyme immobilization. Controlled amounts of PVA-SbQ and of the two enzymes were directly coated on a platinum disk, then photopolymerized. The compatibility of working conditions for choline and phosphatidylcholine detection in the presence of Triton X-100 and CaCl₂ was investigated. The effect of the activity ratio PLaseD / ChOD on the sensor performance was determined. The sensitivities to choline and to phosphatidylcholine were 18 mA.1mol^?1 and 0.66 mA.1.mol^?1 respectively, the detection limit being 1.5.10^?8 M for choline and 1.5.10^?6 M for phosphatidylcholine. The linear range extended up to ca. 10^?4 M for choline and ca. 2.10^?5 M for phosphatidylcholine and the response time was close to 30 seconds for choline and ca. 2 min for phosphatidylcholine.     

Thermokinetic Studies on the Activation of Arginase by Glycine

The activation of bovine liver arginase, which catalyzes the hydrolysis of L‐arginine to L‐ornithine and urea, by glycine was studied by thermokinetic methods at 37°C in 40 mmol·L^?1 sodium barbiturate‐HCl buffer solution (pH 9.4). Results of this experiment indicate that an appropriate concentration of glycine can enhance the activity of arginase, and the relative activation rate reached its maximum value, 74%, when the concentration of glycine in reaction system was 1 mmol·L^?1 and the initial concentration of arginine was 5 mmol·L^?1. With the increase of substrate concentration, the relative activation rate decreased in a definite glycine concentration. Michealis constant K_m of reaction decreased from 5.53 to 3.31 mmol·L^?1 and inhibition constant of product L‐ornithine K_p increased from 1.18 to 3.73 mmol·L^?1 when glycine concentration was 1 mmol·L^?1. For these reasons one possible activation mechanism of arginase by glycine was suggested that the activation effect results from the competition of glycine and arginine to enzyme activity position. When one or two of the activity positions of arginase are occupied by glycine, it is propitious for the enzyme to complex with substrate and obstruct L‐ornithine from combining with enzyme, and when all of the activity positions are occupied by glycine, the activation effect vanishs and the inhibition effect appears.     

Purification of chloroperoxidase from Musa paradisiaca stem juice

Chloroperoxidase from Musa paradisiaca stem juice has been purified to homogeneity using a concentration obtained by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The purified enzyme gave a single protein band in SDS‐PAGE analysis corresponding to molecular mass of 43 kDa. The native PAGE analysis result has also given a single protein band, confirming the purity of the enzyme. The purified enzyme was chlorinated and brominated with monochlorodimedone, the substrate used for measuring the halogenating activity of chloroperoxidases. The K_m and k_cat values using monochlorodimedone as the substrate were 20 μM and 1.64 s^?1, respectively, giving a k_cat/K_m value of 8.2 × 10⁴ M^?1 s^?1. The pH and temperature optima of the chlorinating activity were 3.0 and 25°C, respectively. The K_m values for the peroxidase activity using pyragallol and H₂O₂ as the variable substrates were 89 and 120 μM, respectively. The pH and temperature optima of the peroxidase activity using pyrogalllol as the substrate were the same as the pH and temperature optima of the halogenating activity. The peroxidase activity of the enzyme is competitively inhibited by sodium azide, indicating that it is a hemeperoxidase different from nonheme peroxidases. © 2012 Wiley Periodicals, Inc. Int J Chem Kinet 45: 92–100, 2013     

Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 μM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e⁻/H⁺ acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.     

A Bottom‐Up Proteomic Approach to Identify Substrate Specificity of Outer‐Membrane Protease OmpT

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer‐membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1′) and nearest‐neighbor positions (P2, P2′) and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400‐fold improvement in OmpT catalytic efficiency, with a k _cat/K _m value of 6.1×10⁶ L mol⁻¹ s⁻¹. Wild‐type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.     

Improved Cyclopropanation Activity of Histidine‐Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilnacipran

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative‐scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active‐site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450‐BM3), a highly active His‐ligated variant of P450‐BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3‐Hstar, catalyzes the cyclopropanation of N,N‐diethyl‐2‐phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non‐natural enzyme activity.     

What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

The nonheme iron enzyme OrfP reacts with l -Arg selectively to form the 3R,4R-dihydroxyarginine product, which in mammals can inhibit the nitric oxide synthase enzymes involved in blood pressure control. To understand the mechanisms of dioxygen activation of l -Arg by OrfP and how it enables two sequential oxidation cycles on the same substrate, we performed a density functional theory study on a large active site cluster model. We show that substrate binding and positioning in the active site guides a highly selective reaction through C³−H hydrogen atom abstraction. This happens despite the fact that the C³−H and C⁴−H bond strengths of l -Arg are very similar. Electronic differences in the two hydrogen atom abstraction pathways drive the reaction with an initial C³−H activation to a low-energy ⁵σ-pathway, while substrate positioning destabilizes the C⁴−H abstraction and sends it over the higher-lying ⁵π-pathway. We show that substrate and monohydroxylated products are strongly bound in the substrate binding pocket and hence product release is difficult and consequently its lifetime will be long enough to trigger a second oxygenation cycle.     

Purification and characterization of a microbial dehydrogenase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M _r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD⁺ less effectively than NADP⁺. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD⁺ or NADP⁺. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl₂. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD⁺ and NADP⁺ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD⁺ or NADP⁺. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.     

Bilirubin-sensitized photoinactivation of enzymes in the isolated membrane of the human erythrocyte.

Abstract— Bilirubin has been found to sensitize the photodynamic inactivation of several enzymes in the isolated membrane (ghost) of the human red cell. When ghosts (pH 8.0, 10°C) + bilirubin (0.1 mM) were irradiated with blue light (350 Wm^-2), the activity of glyceraldehyde 3-phosphate dehydrogenase decayed with t_1/2? 15 min. No effect was observed in the absence of pigment or with incident yellow light. Diazabicyclo-octane (DABCO) sharply reduced the inactivation rate, suggesting that ¹O₂ is involved. Sodium dodecyl sulfate-gel electrophoresis of ghosts containing fully inactivated glyceraldehyde 3-phosphate dehydrogenase revealed no change in the polypeptide band corresponding to the subunit of the enzyme. Solubilized enzyme, which was similarly photosensitive, could be partially protected by nicotinamide adenine dinucleotide or glyceraldehyde 3-phosphate. The integral enzymes Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and acetylcholinesterase were also affected. Under the above conditions and bilirubin = 0.37 mM, these enzymes were photoinactivated in first-order fashion, k? 2, 1.2 and 0.2 h^-1, respectively. The rate of decay of total ATPase was found to vary as the square root of the bilirubin concentration over the range 7–370 μM. At a fixed bilirubin concentration (0.37 mM), this rate was also shown to be directly proportional to light intensity. Inasmuch as the —SH content of bilirubin-containing ghosts diminished during irradiation, oxidation of essential cysteine residues could be responsible for the inactivation of some of the enzymes studied.     

Specific Detection and Imaging of Enzyme Activity by Signal‐Amplifiable Self‐Assembling 19F MRI Probes

Specific turn‐on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal‐amplifiable self‐assembling ¹⁹F NMR/MRI probe for turn‐on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are “silent” when aggregated, but exhibit a disassembly driven turn‐on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these ¹⁹F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by ¹⁹F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by ¹⁹F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn‐on ¹⁹F probes may serve as a screening platform for the activity of MMPs.     

Comparative molecular chemistry of molybdenum and tungsten and its relation to hydroxylase and oxotransferase enzymes

The similarities and differences in the fundamental coordination chemistry of molybdenum and tungsten mainly in physiological oxidation states M^IV–VI are examined in relation to the properties of enzyme sites that catalyze oxygen atom transfer reactions. The comparative aspects of dithiolene complexes, which as synthetic analogues simulate structural and electronic features of these sites, are emphasized. Analogue reaction systems of enzymes are summarized. The mechanism of reduction of the biological substrate Me₂SO in one such system as elucidated with density functional calculations is presented as a case study.     

Regioselective Dichlorination of a Non‐Activated Aliphatic Carbon Atom and Phenolic Bismethylation by a Multifunctional Fungal Flavoenzyme

The regioselective functionalization of non‐activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono‐ and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O‐methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH₂‐dependent enzyme is uniquely capable of the stepwise gem‐dichlorination of a non‐activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.