The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine.

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.     

饲料中添加中性蛋白酶对青鱼生长、消化及鱼体组成的影响

用含中性蛋白酶8000U/g的酶制剂,制作酶制剂使用量分别为0‰、0.5‰、1‰、2‰和3‰的5种实验饲料,喂养平均初始重为(3.03±0.04)g的5组三重复的青鱼鱼种8周。实验在容积为80L的15只循环式水族箱中进行。实验水温为(25.0±0.5)℃。结果表明：实验饲料中酶制剂含量为0.5‰时,实验鱼的鱼体增重与摄食不含酶制剂饲料的实验组无显著差异(P>0.05), 而当饲料中酶制剂含量提高至1‰时,鱼体增重出现显著提高(P<0.05)。但饲料中酶制剂含量从1‰进一步提高至2‰和3‰,则鱼体增重不再有显著变化(P>0.05);饲料中酶制剂含量为1‰、2‰和3‰实验组的摄食量显著高于饲料中酶制剂含量为0和0.5‰的实验组(P<0.05)。而饲料中酶制剂含量为1‰、2‰和3‰实验组的饲料系数显著低于饲料中酶制剂含量为0和0.5‰的实验组(P<0.05);青鱼饲料中使用酶制剂对饲料干物质的表观消化率、实验鱼的肠蛋白酶活性、肠和肝胰脏淀粉酶活性、全鱼营养组成、脏体指数、肝体指数无显著影响(P>0.05);饲料中0.5‰－3‰酶制剂含量时,实验鱼对饲料蛋白质的表观消化率均显著高于饲料中不含酶制剂的实验组 (P<0.05)。青鱼鱼种肝胰脏蛋白酶活性随着饲料中酶制剂使用量从0增加0.5‰和1‰而呈逐渐升高的趋势,但饲料中酶制剂使用量继续增加则不再有显著变化。因此,在本实验条件下,饲料中使用1‰－3‰的中性蛋白酶制剂促进青鱼鱼种的生长和降低饲料系数。     

A new approach for dynamics of enzyme-catalyzed glutathione conjugation by electrospray quadrupole/time-of-flight mass spectrometry.

The dynamics of enzyme-catalyzed glutathione conjugation was studied by electrospray quadrupole/time-of-flight (Q-TOF) mass spectrometry with a nanospray interface. After incubation of human glutathione S-transferase A1-1 (GT) with glutathione (GSH) and an electrophilic substrate, electrospray indicated the presence of enzyme/product adducts such as [2GT + product], [2GT + GSH' + product], and [2GT + 2 products] as well as [2GT] and [2GT + GSH']. The relative abundance of GT/product adduct ions increased with incubation time. The wide m/z range of detection (m/z 300-5000) allowed the observation of product, suggested to be released from enzyme/product adducts, in the same mass spectrum. The noncovalent complexes of GT/product were completely replaced by GT/inhibitor complexes following the addition of GT inhibitor to the incubation mixture. Furthermore, a collision-activated decomposition analysis of these ion species provided us with useful information to interpret or identify ion species. The results suggest that electrospray Q-TOF mass spectrometry is a powerful approach for studying the dynamics of the enzyme reaction as well as the structure of enzyme complexes at high sensitivity.     

New method for the histochemical demonstration of dipeptidyl aminopeptidase I activity using a novel anthraquinoyl hydrazide substrate.

A new method for the histochemical localization of dipeptidyl aminopeptidase I (DPP I, cathepsin C), based on a newly synthesized substrate-Gly-L-Phe-5-chloro-1-anthraquinoyl hydrazide.HCl (Gly-Phe-CAH), is proposed. The enzyme activity liberates 5-chloro-1-anthraquinoyl hydrazine (CAH)--a water-insoluble brown-reddish compound, which precipitates on the enzyme locations. The primary reaction product reacts simultaneously or, otherwise, by post-coupling with p-anisaldehyde (p-AA), thus converting to the reddish-violet amorphous hydrazone--the final reaction product. The validity of enzyme localization is thus assured by the insolubility of the primary reaction product and does not depend on the rate of the second reaction step. The enzyme studied is successfully localized in different rat organs using the newly proposed technique.     

Sphingosine kinase 1 is an intracellular effector of phosphatidic acid

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK1 is of considerable interest. We report that SK1, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK1 with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK1-PA interaction maps to the C terminus of SK1 and is independent of catalytic activity or of the diacylglycerol kinase-like domain of the enzyme. Thus SK1 constitutes a novel, physiologically relevant PA effector.     

Measurement of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase activity in human tissues.

The activity of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was measured in normal and diseased human liver, brain and kidney. Organs from patients with aspartylglucosaminuria show very little activity. Crude homogenates of human organs show a reaction catalysed by a complex enzyme system. With homogenate, the formation of product was linear with time up to about 6 h. Reaction times longer than 6-7h resulted in a decrease in the total concentration of product. This phenomenon was not found with the partially purified enzyme fraction. Linearity of the enzyme activity with different protein concentrations was found, independent of the incubation time. Longer incubation of the crude homogenate resulted in the utilization of the product, N-acetylglucosamine. This phenomenon was not observed with the partially purified enzyme fraction. This amidase from human organs differs from that obtained from other sources and apparently represents a rather complex enzyme system.     

Light product of photoreactive 6-azido-FAD bound to deflavo-milk xanthine oxidase.

Xanthine oxidase from milk was reconstituted with the photoreactive flavin, 6-azido-FAD. While irradiation of the reconstituted enzyme under anaerobic conditions yielded 6-amino-FAD as a light product, aerobic irradiation resulted in formation of an unknown product, which gave the enzyme almost the same activity as that of the native enzyme. The light product could be extracted from the enzyme without breakdown and was found to be highly fluorescent. Upon treatment with phosphodiesterase, this light product was converted to the FMN form. The absorption spectrum of the FMN form has a peak at 464 nm, a shoulder at 450 nm in the visible region, and two peaks at 260 and 298 nm in the UV. Irradiation of free 6-azido-3-methyllumiflavin in the presence of a saturating concentration of oxygen yielded a light product whose absorbance and fluorescence spectra were very similar to those of the light product extracted from the enzyme, suggesting that the two had undergone some common photochemical change at the same place in the isoalloxazine ring. Analysis of the light product of 6-azido-3-methyllumiflavin with 1H NMR and FAB mass spectrometry suggested its possible structure with a new five-membered ring, C(6) = N-O-CH = C(7), adjacent to the benzene ring of the flavin.     

Insight into Temperature Dependence of GTPase Activity in Human Guanylate Binding Protein-1

Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.     

Product catalyzes the deamidation of D145N dehalogenase to produce the wild-type enzyme

Aspartate 145 plays an essential role in the active site of 4-chlorobenzoyl-CoA dehalogenase, forming a transient covalent link at the 4-position of the benzoate during the conversion of the substrate to 4-hydroxybenzoyl-CoA. Replacement of Asp 145 by residues such as alanine or serine results in total inactivation, and stable complexes can be formed with either substrate or product. The Raman spectroscopic characterization of some of the latter is described in the preceding publication (Dong et al.). The present work investigates complexes formed by D145N dehalogenase and substrate or product. Time-resolved absorption and Raman difference spectroscopic data show that these systems evolve rapidly with time. For the substrate complex, initially the absorption and Raman spectra show the signatures of the substrate bound in the active site of the asparagine 145 form of the enzyme but these signatures are accompanied by those for the ionized product. After several minutes these signatures disappear to be replaced with those closely resembling the un-ionized product in the active site of wild-type dehalogenase. Similarly, for the product complex, the absorption and Raman spectra initially show evidence for ionized product in the active site of D145N, but these are rapidly replaced by signatures closely resembling the un-ionized product bound to wild-type enzyme. It is proposed that product bound to the active site of asparagine 145 dehalogenase catalyzes the deamidation of the asparagine side chain to produce the wild-type aspartate 145. For the complexes involving substrate, the asparagine 145 enzyme population contains a small amount of the WT enzyme, formed by spontaneous deamidation, that produces product. In turn, these product molecules catalyze the deamidation of Asn 145 in the major enzyme population. Thus, conversions of substrate to product and of D145N to D145D dehalogenase go on simultaneously. The spontaneous deamidation of asparagine 145 has been characterized by allowing the enzyme to stand at RT in Hepes buffer at pH 7.5. Under these conditions deamidation occurs with a rate constant of 0.0024 h-1. The rate of product-catalyzed deamidation in Hepes buffer at 22 degrees C was measured by stopped-flow kinetics to be 0.024 s-1, 36000 times faster than the spontaneous process. A feature near 1570 cm-1 could be observed in the early Raman spectra of both substrate and product-enzyme complexes. This band is not associated with either substrate or product and is tentatively assigned to an ester-like species formed by the attack of the product's 4-O- group on the carbonyl of asparagine's side chain and the subsequent release of ammonia. A reaction scheme is proposed, incorporating these observations.     

Laser excitation studies of the product release steps in the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase

The latter stages of the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6-ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP+ and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP+ is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s(-1). The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP+ concentration, suggesting that conformational changes are required prior to NADP+ release. Following NADP+ release, the NADPH rebinds to the enzyme with a maximum rate constant of approximately 72 s(-1). At elevated temperatures (>298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s(-1). The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.     

Kinetics and mechanism of the rat brain phenol sulphotransferase reaction.

Some properties of rat brain phenol sulphotransferase were investigated in in vitro at pH7.4. The enzyme was purified 10-fold by chromatography on DEAE-Sephadex -50. It can be assayed with 4-hydroxy-3-methoxyphenylethylene glycol or 4-methylumbelliferone as the sulphate acceptor. The partially purified enzyme is stable for at least 1 week when stored at 4 degrees C. It is, however, additionally activated (10--20%) and stabilized by 1 mM-dithiothreitol. The activity of the enzyme does not depend on the addition of exogenous Mg2+. The pH optima for the sulphation of 4-hydroxy-3-methoxyphenylethylene glycol and 4-methylumbelliferone are 7.8 and 7.4 respectively. Substrate inhibition by the sulphate acceptor is apparent at concentrations over 0.05mM. Initial-velocity studies in the absence and presence of product and dead-end inhibitors suggested that the mechanism of the rat brain sulphotransferase reaction is sequential ordered Bi Bi with a dead-end complex of enzyme with adenosine 3',5'-biphosphate and sulphate acceptor. The sulphate donor adenosine 3'-phosphate 5'-sulphatophosphate is the first substrate that adds to the enzyme, and the sulphate acceptor is the second substrate. The dissociation constant for the complex of enzyme with sulphate donor is 21 micron. The sulphated substrate is the first product and adenosine 3',5'-biphosphate is the second product that leaves the enzyme.     

Studies on the equilibria and kinetics of the reactions of ferrous catalase with ligands

The optical absorption spectrum of bovine liver catalase was found to change on light irradiation in the presence of proflavin and EDTA in a deaerated solution. Upon addition of CO to the photolyzed product, the spectrum changed to an another form, suggesting that the photolyzed product is the ferrous form of the enzyme and CO is bound to the ferrous enzyme. When O2 was introduced into the ferrous enzyme, the absorption spectrum returned to its original ferric state. An intermediate spectrum was obtained in this reaction at -20 degrees C in 33% v/v ethylene glycol. Judged from the spectral characteristics of this compound, it is probably an oxyferrous enzyme. It was converted into ferric enzyme gradually when the sample was left at room temperature. The ferrous enzyme, which was generated by flash photolysis of the CO complex of the enzyme in an air-saturated buffer, reacted with O2 to form the oxyferrous enzyme with a second order rate constant of 9.2 x 10(3) M-1.s-1 at pH 8.6 and 20 degrees C. The oxyferrous enzyme thus obtained autodecomposed into the ferric form with a rate constant of 0.1 s-1.     

Identification of a phosphoenolpyruvate:fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes.

Listeria monocytogenes is a gram-positive bacterium whose carbohydrate metabolic pathways are poorly understood. We provide evidence for an inducible phosphoenolpyruvate (PEP):fructose phosphotransferase system (PTS) in this pathogen. The system consists of enzyme I, HPr, and a fructose-specific enzyme II complex which generates fructose-1-phosphate as the cytoplasmic product of the PTS-catalyzed vectorial phosphorylation reaction. Fructose-1-phosphate kinase then converts the product of the PTS reaction to fructose-1,6-bisphosphate. HPr was shown to be phosphorylated by [32P]PEP and enzyme I as well as by [32P]ATP and a fructose-1,6-bisphosphate-activated HPr kinase like those found in other gram-positive bacteria. Enzyme I, HPr, and the enzyme II complex of the Listeria PTS exhibit enzymatic cross-reactivity with PTS enzyme constituents from Bacillus subtilis and Staphylococcus aureus.     

Selectivity of fungal sesquiterpene synthases: role of the active site's H-1 alpha loop in catalysis

Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.     

An acetylenic mechanism-based inhibitor of dopamine beta-hydroxylase

The catalytic action of dopamine beta-hydroxylase on 1-phenyl-1-propyne results in concomitant loss of enzyme activity. At pH 5.5 and 25 degrees C, 1-phenyl-1-propyne inactivates dopamine beta-hydroxylase in a mechanism-based fashion. The inactivation rate is first-order, follows saturation kinetics, and is strictly dependent on catalysis (oxygen and ascorbate are essential). The inactivation rate of saturating 1-phenyl-1-propyne (kinact) increases from 0.08 to 0.22 min-1 when the oxygen saturation increases from 21 to 100%, respectively. Inactivation also requires a copper-containing catalytically competent enzyme. Tyramine and norepinephrine (respectively, substrate and product of the normal catalytic reaction) protect against inactivation, and no regain of enzyme activity occurs after prolonged dialysis. Experiments with ether-extracted incubation solutions (+/- enzyme) showed no difference in their gas chromatography-mass spectral patterns implying that inactivation of dopamine beta-hydroxylase by 1-phenyl-1-propyne occurs through a kinetic process with a partition ratio (kcat/kinact) equal to or near 1. Thus, this acetylenic substrate analog appears to be a very efficient mechanism-based inhibitor of dopamine beta-hydroxylase. We propose that inactivation of this enzyme by 1-phenyl-1-propyne proceeds by formation of a reactive intermediate that occurs prior to product formation and that alkylates an amino acid residue at the active site of the enzyme.     

Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study

The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.     

Metabolism of D-arabinose: origin of a D-ribulokinase activity in Escherichia coli

The kinase responsible for the phosphorylation of d-ribulose was purified 45.5-fold from a strain of Escherichia coli K-12 capable of growth on d-arabinose with no separation of d-ribulo- or l-fuculokinase activities. Throughout the purification, the ratios of activities remained essentially constant. A nonadditive effect of combining both substrates in an assay mixture; identical K(m) values for adenosine triphosphate with either l-fuculose or d-ribulose as substrate; and, the irreversible loss of activity on both substrates, after removal of magnesium ions from the enzyme preparation, suggest that the dual activity is due to the same enzyme. A fourfold greater affinity of the enzyme for l-fuculose than for d-ribulose, as well as a higher relative activity on l-fuculose, suggest that the natural substrate for this enzyme is l-fuculose. The product of the purified enzyme, with d-ribulose as substrate, was prepared. The ratio of total phosphorous to ribulose phosphate was 1.01:1, indicating that the product was ribulose monophosphate. The behavior of the kinase product in the cysteine-carbazole and orcinol reactions, as well as the results of periodate oxidation assays, provided evidence that it was not d-ribulose-5-phosphate. Reaction of this compound with a cell-free extract of E. coli possessing l-fuculose-l-phosphate aldolase activity resulted in the production of dihydroxyacetone phosphate and glycolaldehyde. The kinase product failed to reduce 2,3,5-triphenyltetrazolium and possessed a half-life of approximately 1.5 min in the presence of 1 n HCl at 100 C. These properties suggested that the phosphate group was attached to carbon atom 1 of d-ribulose.     

Co(II)-regulated substrate specificity of cytosolic alpha-mannosidase

Cytosolic neutral alpha-mannosidase is a putative catabolic enzyme that produces cytosolic free oligomannosides. Activation of the enzyme by Co(II) treatment has been reported using pyridylamino derivatives of Man(5)GlcNAc and Man(5)GlcNAc2, and p-nitrophenyl alpha-mannoside as substrates, with the Co(II)-treated enzyme releasing four alpha-mannose residues from Man(9)GlcNAc to give Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc as an end product. When Man(9)GlcNAc, which is considered to be the actual substrate in the cytosol, was used as a substrate, we found that even before treatment with Co(II) the enzyme was able to cleave a single Manalpha1-2 residue from Man(9)GlcNAc to give Manalpha1-6(Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc as the end product. The K(m) value of the Co(II)-treated enzyme for Man(9)GlcNAc was found to be 37 microM, which is one-twelfth that of the non-treated enzyme, while the values were V(max) values were almost the same, indicating that the affinity of the substrate is higher with Co(II). These results indicate that Co(II) regulates the substrate specificity of the enzyme.     

Cyanide formation from glyoxylate and hydroxylamine catalysed by extracts of higher-plant leaves

Extracts of spinach, maize and barley contain an enzyme which catalyses the formation of hydrogen cyanide from glyoxylate and hydroxylamine. The enzyme is dependent upon ADP and a divalent cation such as manganese. Glyoxylicacid oxime is a poor substrate for the enzyme. Carbon dioxide is another product of the reaction and is probably produced in 1:1 stoichiometry with hydrogen cyanide. The possible relationship of this enzyme to the regulation of nitrate reduction is discussed.     

Kinetic mechanism of human apurinic/apyrimidinic endonuclease action in nucleotide incision repair

Human major apurinic/apyrimidinic endonuclease (APE1) is a multifunctional enzyme that plays a central role in DNA repair through the base excision repair (BER) pathway. Besides BER, APE1 is involved in an alternative nucleotide incision repair (NIR) pathway that bypasses glycosylases. We have analyzed the conformational dynamics and the kinetic mechanism of APE1 action in the NIR pathway. For this purpose we recorded changes in the intensity of fluorescence of 2-aminopurine located in two different positions in a substrate containing dihydrouridine (DHU) during the interaction of the substrate with the enzyme. The enzyme was found to change its conformation within the complex with substrate and also within the complex with the reaction product, and the release of the enzyme from the complex with the product seemed to be the limiting stage of the enzymatic process. The rate constants of the catalytic cleavage of DHU-containing substrates by APE1 were comparable with the appropriate rate constants for substrates containing apurinic/apyrimidinic site or tetrahydrofuran residue, which suggests that NIR is a biologically important process.