Monamine oxidase in outer membrane of skeletal muscle mitochondria.

(1) Monoamine oxidase (EC 1.4.3.4) is present in rat skeletal muscle mitochondria. (2) A radioassay procedure for the assay of monoamine oxidase in muscle mitochondria is described. It is based on teh procedure using side-chain [2-14C]-tryptamine as substate described by Wurtman, R.J. and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439--1441 and employs a pH of 8.0 and a substrate concentration of 0.25 mM. (3) The Km of the muscle mitochondrial enzyme at pH 8.0 is 1.34 - 10(-5) M and that of the liver enzyme under the same conditions is 2.5 - 10(-5) M. Muscle mitochondria contain only one quarter of the activity of enzyme present in liver mitochondria. (4) Monoamine oxidase is shown to be in the outer membrane of skeletal muscle mitochondria and thus to be a suitable marker enzyme for use in the fractionation of these mitochondria.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. I. Substrate identity

A detailed steady-state kinetic investigation of the hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase is reported. The activity was studied in the presence of (i) Na+ (130 mM), K+ (20 mM) and micromolar ATP concentrations and Na+ (150 mM) the ('Na+-enzyme'). The data obtained lead to the following results: 1. The action of each enzyme may be described by a simple kinetic mechanism with one (Na+-enzyme) or two ((Na+ + K+)-enzyme) dead-end Mg complexes. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg2+, in the latter case, as the second substrate. 3. For each enzyme, the complete set of kinetic constants (seven for the Na+-enzyme, eight for the (Na+ + K+)-enzyme) are determined from the data. 4. For each enzyme it is shown that, in the alternate substrate mechanism obtained, the ratio of net steady-state flux along the 'MgATP pathway' to that of the 'ATP-Mg pathway' increases linearly with the concentration of free Mg2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg2+ concentrations the alternate substrate mechanism degenerates into a 'limiting' kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg2+ as an uncompetitive (Na+-enzyme) or non-competitive ((Na+ + K+)-enzyme) inhibitor.     

The alpha-chymotryptic ydrolysis of glycine esters

1. The alpha-chymotrypsin-catalysed hydrolysis of N-acetylglycine ethyl and thiolethyl esters was investigated at pH7.90 and 25 degrees over a wide range of substrate concentrations. 2. The Lineweaver-Burk plots for these substrates are markedly curved, and it is shown that the curvature is due solely to the ;enzyme-blank' reaction. The rate of this reaction is proportional to free enzyme concentration in the range 10-100mum, with a pseudo-first-order rate constant of approx. 1x10(-3)sec.(-1). Correction for this reaction by the procedure described leads to linear plots. It is shown that the significance of the enzyme-blank reaction depends on the value of k(0)/K(m) for the substrate under investigation. 3. Interpretation of the curvature in the Lineweaver-Burk plots by previous workers in terms of activation by excess of substrate is shown to be erroneous. 4. Values of K(m) 387mm and k(0) 0.039sec.(-1), and K(m) 41mm and k(0) 0.23sec.(-1), were obtained for the ethyl and thiolethyl esters of N-acetylglycine respectively. The literature values for the methyl esters of N-acetyl- and N-propionyl-glycine have been corrected by the procedure described. The new values agree much better with current theories of alpha-chymotrypsin mechanism and specificity. 5. The kinetic parameters for the ethyl and thiolethyl esters indicate the absence of an electrophilic component in the catalytic mechanism of alpha-chymotrypsin, and the importance of the ester function in substrate binding.     

Effect of error of the quasi-steady-state approximation on the estimation of Km and Vm from a single time curve

For an irreversible, one-substrate enzyme mechanism, post-transient time curves of the substrate and the product are approximately described by different equations of the steady-state type. The magnitude of error of these approximations is shown to be small either at low enzyme/substrate or at low enzyme/ Michaelis-constant ratios. The effect of error on the kinetic parameters estimated from a single time curve is evaluated. It is shown that a set of well-separated substrate con centrations (which are still high relative to the concentration of enzyme) is crucial for obtaining accurate estimates of the parameters.     

The mechanism of the synthesis of enzymes. II. Further observations with particular reference to the linear nature of the time course of enzyme formation

1. The pretreatment induction method of studying the formation of beta-galactosidase in E. coli B has been described. 2. It has been found that E. coli B cells have their maximum capacity to form beta-galactosidase, in response to a constant induction stimulus, when they are in the stationary phase of the growth cycle. 3. The concentration of inductor, the nature of the nitrogen source, the duration of the assimilatory phase, oxygen tension, and temperature are factors which affect, and may limit, the rate of beta-galactosidase formation. 4. When limitations imposed by these factors were removed, the time course of induced beta-galactosidase formation was strictly linear from the onset. 5. The implications of this finding were discussed and a new theory of the mechanism of enzyme formation has been proposed. 6. A very satisfactory method of synthesis of ortho-nitrophenol-alpha-D-galactoside has been described. This substance is a suitable chromogenic substrate for the specific determination of alpha-galactosidase activity. 7. Preliminary experiments using this substrate have confirmed the results of respiration studies and shown that in E. coli B alpha-galactosidase formation may be induced by beta- as well as by alpha-galactosides.     

Efficient hydrolysis of hemicellulose by a Fusarium graminearum xylanase blend produced at high levels in Escherichia coli

A Fusarium graminearum-based enzyme blend for the efficient hydrolysis of hemicellulose, a crucial step for competitive bioethanol production, is described. The heretofore-uncharacterized endo-1,4-beta-xylanase (XylD), 1,4-beta-xylosidase (XyloA), and bifunctional xylosidase/arabinofuranosidase (Xylo/ArabA) were produced at high levels in Escherichia coli (10-38 mg/l). They displayed compatible pH and temperature-dependences, allowing their utilization for simultaneous substrate digestions. Monosaccharide analysis indicated a strong positive synergism between the enzymes during the degradation of oat spelt xylan. Two units of each protein catalyzed the release of 61% and 15% of the total amount of available d-xylose and l-arabinose, respectively, in only 4 h. The detailed cooperative mechanism of the three hydrolases was elucidated by polysaccharide analysis using carbohydrate gel electrophoresis (PACE) and the enzymes were shown to be suitable for the partial hydrolysis of pretreated crude plant biomass.     

Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity

Proteoliposome vesicles containing apoA-I, lecithin, and cholesterol (including labeled cholesterol) were prepared from various molar ratios of the three components by the cholate dialysis technique. Comparative studies on the sensitivity and efficiency of these proteoliposomes to serve as substrate for lecithin:cholesterol acyltransferase (LCATase) indicated that the proteoliposome with apoA-I:lecithin:cholesterol molar ratio of 0.8:250:12.5 was ideal for assaying LCATase activity of both plasma and purified enzyme. This proteoliposome was shown to be comparable in size by gel filtration (radius, 131.9 +/- 4.8 A, n = 6) and by electron microscopy (radius, 123.4 +/- 5.1 A, n = 100). The proteoliposome preparation was stable as LCATase substrate for at least 3 and 5 weeks, respectively, when stored at 4 degrees C and -20 degrees C, and was a better substrate for the enzyme activity assay than were lecithin-cholesterol liposomes incubated with apoA-I. Under the standardized assay system LCATase activity was a linear function of plasma enzyme added and was independent of the amount of plasma cholesterol added to the proteoliposomes in the range of 3 to 20 microliters of plasma. The mean LCATase activity by this method was 95.1 +/- 14.0 (range 76.5-122.5) nmol/hr per ml of plasma from fifteen normal human subjects. 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 of LCATase reaction.     

PnrA, a new nitroreductase-family enzyme in the TNT-degrading strain Pseudomonas putida JLR11

Nitroreductases are a group of proteins that catalyse pyridine nucleotide-dependent reduction of nitroaromatics compounds, showing significant human health and environmental implications. In this study we have identified the nitroreductase-family enzymes PnrA and PnrB from the TNT-degrading strain Pseudomonas putida. The enzyme encoded by the pnrA gene was expressed in Escherichia coli, purified to homogeneity and shown to be a flavoprotein that used 2 mol of NADPH to reduce 1 mol of 2,4,6-trinitrotoluene (TNT) to 4-hydroxylamine-2,6-dinitrotoluene, using a ping-pong bi-bi mechanism. The PnrA enzyme also recognized as substrates as a number of other nitroaromatic compounds, i.e. 2,4-dinitrotoluene, 3-nitrotoluene, 3- and 4-nitrobenzoate, 3,5-dinitrobenzamide and 3,5-dinitroaniline expanding the substrates profile from previously described nitroreductases. However, TNT resulted to be the most efficient substrate examined according to the Vmax/Km parameter. Expression analysis of pnrA- and pnrB-mRNA isolated from cells growing on different nitrogen sources suggested that expression of both genes was constitutive and that its level of expression was relatively constant regardless of the growth substrate. This is in agreement with enzyme-specific activity determined with cells growing with different N-sources.     

Quantum chemical modeling of enzymatic reactions: the case of 4-oxalocrotonate tautomerase

The reaction mechanism of 4-oxalocrotonate tautomerase (4-OT) is studied using the density functional theory method B3LYP. This enzyme catalyzes the isomerisation of unconjugated alpha-keto acids to their conjugated isomers. Two different quantum chemical models of the active site are devised and the potential energy curves for the reaction are computed. The calculations support the proposed reaction mechanism in which Pro-1 acts as a base to shuttle a proton from the C3 to the C5 position of the substrate. The first step (proton transfer from C3 to proline) is shown to be the rate-limiting step. The energy of the charge-separated intermediate (protonated proline-deprotonated substrate) is calculated to be quite low, in accordance with measured pKa values. The results of the two models are used to evaluate the methodology employed in modeling enzyme active sites using quantum chemical cluster models.     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

The kinetics of a purified form of 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (tryptophan) from Neurospora crassa.

1. A method is described for the purification of a form of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) that probably differs from that of the native enzyme. 2. The kinetics of the reaction catalysed by 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) shows that the reaction proceeds via a ping-pong bi-bi mechanism, with activation by phosphoenolpyruvate (P-Prv), the first substrate, and inhibition by erythrose 4-phosphate (Ery-P) the second substrate. At low substrate concentrations, KP-Prv is 0.1 mM and KEry-P is 0.13 mM. 3. The substrates phosphoenolpyruvate and erythrose 4-phosphate and the product inorganic phosphate can protect the purified enzyme against heat denaturation, whereas the inhibitor, tryptophan, has no effect, although it binds to the enzyme in the absence of other ligands. 4. Product inhibition by inorganic phosphate is linear non-competitive with respect to phosphoenolpyruvate (Ki, slope = 22 mM and Ki, intercept = 54 mM) and substrate-linear competitive with respect to erythrose 4-phosphate (Ki, slope = 25 mM). 5. The enzyme has an activity optimum at pH 7.3 and a tryptophan inhibition optimum at pH 6.4, Trp 0.5 is 4 microM. Inhibition by tryptophan is non-competitive with respect to phosphoenolpyrovate and substrate-parabolic competitive with respect to erythrose 4-phosphate. 6. The role of the enzyme in metabolic regulation is discussed.     

Hydrolysis of acetals and approach to the modeling mechanism of reactions involving lysozyme]

Using quantum chemistry CNDO/2 method the mechanism of reaction of polysaccharides with lysozyme was investigated. The molecule of acetal (H3C-O-CH2-O-CH3) was taken as the simplest substrate model. In the framework of the simple model the influence of interaction of the substrate with Glu-35 and Asp-52 on activation of the substrate is described. It is essential that for the maximum activation of the bond broken the optimum (but not the most energetically advantageous) arrangement of Glu-35 should be realized. The optimum arrangement of the amino acid residues of the enzyme should also be realized for the liberation of the groups which took part in the reaction, only one degree of freedom being actual in this process, and the motion of the system occurs along this degree of freedom. It was shown that substrate distortion could cause its activation.     

Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate

1. The kinetics of the reaction of di-(2-chloroethyl) 3-chloro-4-methylcoumarin-7-yl phosphate (haloxon) and related compounds with acetylcholinesterase were studied and found to be unusual. 2. By a progressive reaction haloxon produces a di-(2-chloroethyl)phosphorylated enzyme. The influence of substrate on this reaction leading to a phosphorylated active centre was studied. From competition experiments between inhibitor and substrate values of K(m) for acetylcholine and acetylthiocholine of 0.79mm and 0.23mm respectively were derived. 3. Haloxon also combines with acetylcholinesterase by a non-progressive reaction, producing a complex that is reversible by dilution and by high concentrations of acetylcholine and acetylthiocholine. From this non-progressive reaction the competition between haloxon and substrate was studied, and it was shown that haloxon combines with a site involved in inhibition by substrate. From competition experiments the following dissociation constants were derived: for combination of haloxon and this site K(i) is 4.9mum and for the combination of substrates with this site K(88) values are 12mm and 3.3mm for acetylcholine and acetylthiocholine respectively. 4. The non-phosphorus-containing compound 3-chloro-7-hydroxy-4-methylcoumarin was shown to be a good reagent for the site involved in inhibition by substrate; its dissociation constant for the combination with this site is 30mum. 5. In order to interpret the experimental results, theoretical equations were derived for an enzyme with two binding sites to both of which substrate and inhibitor can combine. The equations correlate the activity of the enzyme with the concentration of substrate and inhibitor, for both progressive and non-progressive inhibition. These equations are applicable to reactions of acetylcholinesterase with organophosphorus compounds, carbamates etc. and may be applicable to other enzymes possessing two binding sites.     

The kinetics and mechanism of liver alcohol dehydrogenase with primary and secondary alcohols as substrates

1. The activity of liver alcohol dehydrogenase with propan-2-ol and butan-2-ol has been confirmed. The activity with the corresponding ketones is small. Initial-rate parameters are reported for the oxidation of these secondary alcohols, and of propan-1-ol and 2-methylpropan-1-ol, and for the reduction of propionaldehyde and 2-methylpropionaldehyde. Substrate inhibition with primary alcohols is also described. 2. The requirements of the Theorell-Chance mechanism are satisfied by the data for all the primary alcohols and aldehydes, but not by the data for the secondary alcohols. A mechanism that provides for dissociation of either coenzyme or substrate from the reactive ternary complex is described, and shown to account for the initial-rate data for both primary and secondary alcohols, and for isotope-exchange results for the former. With primary alcohols, the rapid rate of reaction of the ternary complex, and its small steady-state concentration, result in conformity of initial-rate data to the requirements of the Theorell-Chance mechanisms. With secondary alcohols, the ternary complex reacts more slowly, its steady-state concentration is greater, and therefore dissociation of coenzyme from it is rate-limiting with non-saturating coenzyme concentrations. 3. Substrate inhibition with large concentrations of primary alcohols is attributed to the formation of an abortive complex of enzyme, NADH and alcohol from which NADH dissociates more slowly than from the enzyme-NADH complex. The initial-rate equation is derived for the complete mechanism, which includes a binary enzyme-alcohol complex and alternative pathways for formation of the reactive ternary complex. This mechanism would also provide, under suitable conditions, for substrate activation or substrate inhibition in a two-substrate reaction, according to the relative rates of reaction through the two pathways.     

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12. 2. Kinetic properties.

1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.     

A rapid purification of T4 polynucleotide kinase using Blue Dextran-Sepharose chromatography.

A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.     

Purification and properties of type 1 topoisomerase from chicken erythrocytes: mechanism of eukaryotic topoisomerase action

A simple method for the purification of the major topoisomerase (topoisomerase 1) from chicken erythrocytes is described. Because of the generally repressed state of the chromatin from these nuclei, the heterogeneity of the non-histone proteins is reduced, and it is possible to purify this enzyme from a nuclear extract by a single chromatographic step. The chicken erythrocyte topoisomerase appears to be similar to previously described eukaryotic type I topoisomerases with respect to its physical and enzymological properties. The pattern of intermediate products generated during the action of chicken erythrocyte topoisomerase on a supercoiled closed circular DNA substrate has been examined quantitatively and has been shown to be consistent with a mechanism in which the enzyme closes its substrate DNA molecular after the removal of each superhelical turn and in which dissociation of the enzyme substrate complex may, but does not necessarily, occur after each cycle of the reaction.     

Studies of the catalytic mechanism of an active-site mutant (Y14F) of delta 5-3-ketosteroid isomerase by kinetic deuterium isotope effects

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the conversion of androst-5-ene-3,17-dione to androst-4-ene-3,17-dione by a stereoselective transfer of the 4 beta-proton to the 6 beta-position. The rate-limiting step has been shown to be the concerted enolization of the enzyme-bound substrate comprising protonation of the 3-carbonyl oxygen by Tyr-14 and abstraction of the 4 beta-proton by Asp-38 [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500]. Primary, secondary, solvent, and combined kinetic deuterium isotope effects have been used to investigate the mechanism of the Y14F mutant, which lacks the proton donor and is 10(4.7)-fold less active catalytically than the wild-type enzyme. With [4 beta-D]androst-5-ene-3,17-dione as a substrate in H2O, a lag in product formation is observed which approaches, by a first-order process, the rate observed with protonated substrate. With the protonated substrate in D2O, a burst in product formation is detected by derivative analysis of the kinetic data which approaches the rate observed with the 4 beta-deuterated substrate in D2O. The absence of such lags or bursts with the protonated substrate in H2O or with the 4 beta-deuterated substrate in D2O, as well as the detection of buffer catalysis by phosphate at pH 6.8, indicates that one or more intermediates dissociate from the enzyme and partition to substrate 31.6 times faster than to product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of human collagenases with a hydroxamic acid affinity column

Human collagenase has been isolated from skin fibroblasts and rheumatoid synovium by using an affinity matrix, prepared by coupling Pro-Leu-Gly-NHOH to agarose. Following the methodology described herein, the skin enzyme was isolated in two steps in 76% yield and the synovial enzyme was purified in three steps in 71% yield. Importantly, each enzyme hydrolyzed collagen into 3/4-1/4 cleavage fragments, indicating that a true collagenase had been isolated. The column was specific for the human enzyme since the collagenase from Clostridium histolyticum did not bind. The affinity ligand was designed according to the formalism proposed by Holmquist and Vallee [Holmquist, B., & Vallee, B. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6216] that effective metalloenzyme inhibitors can be synthesized by coupling a suitable metal-coordinating group to a substrate analogue. In this case, the hydroxamic acid probably coordinates to the active-site metal and the Pro-Leu-Gly moiety is similar to the carboxyl side of the cleavage site of collagen, the enzyme's substrate. The IC50 for N-(benzyloxycarbonyl)-Pro-Leu-Gly-NHOH is 4 X 10(-5) M for both enzymes. The affinity chromatographic procedures described here should aid in future studies on vertebrate collagenases.