Infinite Substrate Concentration Research Articles

Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde

In this study, the xanthine oxidase (XO) enzyme was purified by affinity chromatography technique using Sepharose-4B-L-tyrosine-4-aminobenzamidine gel and its immobilization with glutaraldehyde was investigated. Using ammonium sulfate precipitation and affinity gel, xanthine oxidase was purified 643.04-fold in an 11.5% yield. The purity of the enzyme was checked by SDS polyacrylamide gel electrophoresis and a single band around 150 kDa was observed. KM (the Michaelis constant) and VMax (the asymptotic reaction velocity at infinite substrate concentration) of the enzyme were determined at 1.67x10-4 M and 0.56 U/mL.min respectively by using a xanthine compound as a substrate. The in vitro effects of NH4F, NH4Cl, CaCl2, ZnCl2, HgCl2, Hg(NO3)2.H2O compounds and commercially named colchicum dispert, commonly used in the treatment of gout disease in the clinic, were investigated. The IC50 values of compounds showing inhibition effects were determined. Afterward, XO was immobilized with glutaraldehyde. The highest XO activity was observed in the sample of the immobilized enzyme at a rate of 6% glutaraldehyde. The kinetic constants (KM and VMax) of the immobilized enzyme were determined as 5.18x10-4 M and 0.73 U mL-1 min-1 respectively. These values revealed that the catalytic activity of the free enzyme was higher than the immobilized enzyme.

When both Km and Vmax are altered, Is the enzyme inhibited or activated?

Enzyme activators lower Km (the Michaelis constant) and/or raise Vmax (the asymptotic reaction velocity at infinite substrate concentration); conversely, inhibitors raise Km and/or lower Vmax . But what if an effector moves both Km and Vmax in the same direction? Uncompetitive inhibitors, which decrease both Km and Vmax by the same factor, are the most common example of this. A less well-known example occurs often when crowding agents are added to the buffer in order to mimic the environment commonly encountered in vivo. Crowding agents tested on different enzymes have been shown to have every conceivable effect on Km or Vmax , causing them to rise, fall, or stay the same. In this article, a mathematical analysis is presented allowing biochemists to judge whether an effector that causes Km and Vmax to both move in the same direction serves as an inhibitor, an activator, or most surprising, as both. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):446-449, 2019.

CaMKII Autonomy Is Substrate-dependent and Further Stimulated by Ca2+/Calmodulin

A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca(2+)-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active ( approximately 70-100%), implicating that it is no longer regulated and that its dramatically increased Ca(2+)/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15-25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca(2+)/CaM, both in vitro and within cells. Altered K(m) and V(max) made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca(2+) signals, but prevents complete uncoupling from subsequent cellular stimulation.

Kinetic and magnetic resonance studies of the role of metal ions in the mechanism of Escherichia coli GDP-mannose mannosyl hydrolase, an unusual nudix enzyme.

Escherichia coli GDP-mannose mannosyl hydrolase (GDPMH), a homodimer, catalyzes the hydrolysis of GDP-alpha-D-sugars to yield the beta-D-sugar and GDP by nucleophilic substitution with inversion at the C1' carbon of the sugar [Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608]. GDPMH requires a divalent cation for activity such as Mn2+ or Mg2+, which yield similar kcat values of 0.15 and 0.13 s(-1), respectively, at 22 degrees C and pH 7.5. Kinetic analysis of the Mn2+-activated enzyme yielded a K(m) of free Mn2+ of 3.9 +/- 1.3 mM when extrapolated to zero substrate concentration (K(a)Mn2+), which tightened to 0.32 +/- 0.18 mM when extrapolated to infinite substrate concentration (K(m)Mn2+). Similarly, the K(m) of the substrate extrapolated to zero Mn2+ concentration (K(S)(GDPmann) = 1.9 +/- 0.5 mM) and to infinite Mn2+ concentration (K(m)(GDPmann) = 0.16 +/- 0.09 mM) showed an order of magnitude decrease at saturating Mn2+. Such mutual tightening of metal and substrate binding suggests the formation of an enzyme-metal-substrate bridge complex. Direct Mn2+ binding studies, monitoring the concentration of free Mn2+ by EPR and of bound Mn2+ by its enhanced paramagnetic effect on the longitudinal relaxation rate of water protons (PRR), detected three Mn2+ binding sites per enzyme monomer with an average dissociation constant (K(D)) of 3.2 +/- 1.0 mM, in agreement with the kinetically determined K(a)Mn2+. The enhancement factor (epsilon(b)) of 11.5 +/- 1.2 indicates solvent access to the enzyme-bound Mn2+ ions. No cross relaxation was detected among the three bound Mn2+ ions, suggesting them to be separated by at least 10 A. Such studies also yielded a weak dissociation constant for the binary Mn2+-GDP-mannose complex (K1 = 6.5 +/- 1.0 mM) which significantly exceeded the kinetically determined K(m) values of Mn2+, indicating the true substrate to be GDP-mannose rather than its Mn2+ complex. Substrate binding monitored by changes in 1H-15N HSQC spectra yielded a dissociation constant for the binary E-GDP-mannose complex (K(S)(GDPmann)) of 4.0 +/- 0.5 mM, comparable to the kinetically determined K(S) value (1.9 +/- 0.5 mM). To clarify the metal stoichiometry at the active site, product inhibition by GDP, a potent competitive inhibitor (K(I) = 46 +/- 27 microM), was studied. Binding studies revealed a weak, binary E-GDP complex (K(D)(GDP) = 9.4 +/- 3.2 mM) which tightened approximately 500-fold in the presence of Mn2+ to yield a ternary E-Mn2+-GDP complex with a dissociation constant, K3(GDP) = 18 +/- 9 microM, which overlaps with the K(I)(GDP). The tight binding of Mn2+ to 0.7 +/- 0.2 site per enzyme subunit in the ternary E-Mn2+-GDP complex (K(A)' = 15 microM) and the tight binding of GDP to 0.8 +/- 0.1 site per enzyme subunit in the ternary E-Mg2+-GDP complex (K3 < 0.5 mM) indicate a stoichiometry close to 1:1:1 at the active site. The decrease in the enhancement factor of the ternary E-Mn2+-GDP complex (epsilon(T) = 4.9 +/- 0.4) indicates decreased solvent access to the active site Mn2+, consistent with an E-Mn2+-GDP bridge complex. Fermi contact splitting (4.3 +/- 0.2 MHz) of the phosphorus signal in the ESEEM spectrum established the formation of an inner sphere E-Mn2+-GDP complex. The number of water molecules coordinated to Mn2+ in this ternary complex was determined by ESEEM studies in D2O to be two fewer than on the average Mn2+ in the binary E-Mn2+ complexes, consistent with bidentate coordination of enzyme-bound Mn2+ by GDP. Kinetic, metal binding, and GDP binding studies with Mg2+ yielded dissociation constants similar to those found with Mn2+. Hence, GDPMH requires one divalent cation per active site to promote catalysis by facilitating the departure of the GDP leaving group, unlike its homologues the MutT pyrophosphohydrolase, which requires two, or Ap4A pyrophosphatase, which requires three.

Understanding Enzyme Inhibition

While enzyme inhibition is a widely taught subject across chemical and biochemical disciplines, it remains poorly understood. A mental image is presented to facilitate the understanding of inhibition types other than competitive. Subsequently, enzyme inhibition is developed using Vmax/Km in place of Km. Interpretation of direct (initial velocity vs substrate concentration) plots makes clear the meanings of competitive, noncompetitive, and mixed inhibition in a manner entirely distinct from current textbook treatments. The effects of inhibitors on enzymes can be seen to be reduced to a simple consideration of actions at zero and infinite substrate concentrations, corresponding to Vmax/Km and Vmax, respectively.

Limited proteolysis and X-ray crystallography reveal the origin of substrate specificity and of the rate-limiting product release during oxidation of D-amino acids catalyzed by mammalian D-amino acid oxidase.

Limited proteolysis of D-amino acid oxidase holoenzyme with trypsin cleaves the protein at Arg 221 and near the C-terminus, producing stable 25, 13.4, and 2 kDa polypeptides [Torri-Tarelli, G., Vanoni, M. A., Negri, A., & Curti, B. (1990) J. Biol. Chem. 265, 21242-21246]. The 25 and 13.4 kDa polypeptides remain associated to form a nicked D-amino acid oxidase species. This nicked protein form maintains the ability to bind FAD, but exhibits altered catalytic efficiency toward the oxidation of various D-amino acids when compared to native DAAO. Changes in substrate specificity were first monitored by measuring the activity in the presence of different amino acid substrates at various times during proteolysis. Three amino acid substrates were then selected for further analysis of the properties of the nicked D-amino acid oxidase species produced by limited tryptic proteolysis: D-serine, D-arginine, and D-alanine. The three D-amino acids represented limiting cases of the observed changes of enzyme activity on nicking: loss of activity, increase of activity, and minor activity changes, respectively. D-serine was found to be no longer a substrate of D-amino acid oxidase. D-arginine exhibited a 2.5-fold increased apparent maximum velocity although its Km value increased 2-fold with the nicked enzyme in comparison to the native species. D-alanine was oxidized 1.5-fold faster by the nicked D-amino acid oxidase at infinite substrate concentration, and its Km value increased approximately 4-fold. The Kd for benzoate, which was determined kinetically with D-alanine as the enzyme substrate, increased 17-fold in the nicked species. Primary deuterium kinetic isotope effects on V and V/K during the oxidation of D-alanine were also measured. (D)V/K increased from 1.4 +/- 0.2 to 1.8 +/- 0.3 on nicking, while (D)V increased from 1.04 +/- 0.1 to 2.53 +/- 0.5. All the observed changes of the values of the kinetic parameters and of the observed isotope effects are consistent with the hypothesis that nicking of D-amino acid oxidase at position 221 decreases the strength of binding of both substrates and products to the enzyme active site. The information obtained by limited tryptic proteolysis nicely complements that gathered from the analysis of the three-dimensional structure of D-amino acid oxidase in complex with benzoate, which was recently determined [Mattevi, A., Vanoni, M. A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., & Curti, B. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7496-7501]. Arginine 221 is part of the 216-228 loop that covers the active site and contributes residues to substrate binding and catalysis. The limited proteolysis data support the hypothesis that this loop acts as a lid on the active site and controls both substrate specificity and the rate of turnover of D-amino acid oxidase.

Phosphatidylinositol 4,5-Bisphosphate Binding to the Pleckstrin Homology Domain of Phospholipase C-δ1 Enhances Enzyme Activity

The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some co-complexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-delta1 (PLCdelta1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in a dose-dependent manner with an EC50 = 1 microM (0.3 mol%), up to 9-fold higher when 5 microM (1.5 mol%) of PIP2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 microM of PI with a molar ratio of PI:PS:PC = 1:5:5). Stimulation was specific for PIP2, since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited by inositol 1,4, 5-triphosphate (IP3) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLCdelta1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP2-mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP2. Analysis of PIP2-stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (Ks) of PLCdelta1 for the PI/PS/PC vesicles was reduced from 558 microM to 53 microM, and the interfacial Michaelis constant (Km) was reduced from 0.21 to 0.06 by PIP2. The maximum rate of PI hydrolysis (Vmax) was not affected by PIP2. These results demonstrate that a major function of the PH domain of PLCdelta1 is to modulate enzyme activity. Further, our results identify PIP2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP2.

Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis

The reaction of two D-amino acid oxidases from the yeasts Rhodotorula gracilis and Trigonopsis variabilis with the substrates alanine and valine in their 2-1H and 2-2H forms was studied employing the stopped-flow spectrophotometric technique. The turnover numbers at infinite substrate and oxygen concentrations were: 20,700/4,250 and 1,730/360 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,150/440 and 2,500/520 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The rates of anaerobic enzyme flavin reduction were 20,100/4,000 and 1,820/350 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,470/350 and 2,460/480 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The isotope effects on enzyme reduction were 5.0 and 5.2 for Rhodotorula and 9.9 and 5.1 for Trigonopsis D-amino acid oxidases with alanine and valine, respectively. This suggests that the intrinsic isotope effect on rupture of the substrate alpha-C-H bond can be as high as 10. The rate-determining step corresponds to the enzyme reductive half-reaction in contrast to the mammalian kidney enzyme where it is the product release from oxidized enzyme (Massey, V., and Gibson, Q.H. (1964) Fed. Proc. 23, 18-29). Upon anaerobic reaction with substrate, the yeast enzymes do not form the transient long wavelength absorbing species which are characteristic of the mammalian protein. This is due only in part to rapid dissociation of iminoacid product and is ascribed to intrinsic differences between the charge-transfer complexes of reduced enzyme flavin and product of the yeast as compared to the mammalian enzyme. With the Trigonopsis enzyme the flavin radical anion appears to be strongly stabilized and can be produced quantitatively.

Recombinant type A rat 75-kDa α-amidating enzyme catalyzes the conversion of glycine-extended peptides to peptide amides via an α-hydroxyglycine intermediate

The amidation of C-terminal glycine-extended peptides has been analyzed by the use of a truncated type A peptidylglycine α-amidating enzyme (α-AE) encoded by cDNA prepared with RNA from rat medullary thyroid carcinoma (MTC) cells. Mouse C127 cells transfected with the rat MTC cDNA encoding the truncated type A α-AE secrete the expected 75-kDa enzyme into the culture medium. Medium conditioned with the transfected C127 cells converts both dansyl-TyrValGly and dansyl-TyrValα-hydroxyglycine to dansyl-TyrValNH 2 at levels which are approximately 1000 times higher than the levels found in medium conditioned with untransfected C127 cells. This result indicates that rat type A α-AE alone catalyzes a two-step reaction involving an initial hydroxylation of peptidyl-Gly followed by conversion of the peptidyl-α-hydroxyglycine intermediate to the amidated product. The involvement of a separate, second enzyme to convert peptidyl-α-hydroxyglycine to peptidyl-NH 2 is not necessary in this system. The initial hydroxylation step is rate-determining at infinite substrate concentration and requires a reducing equivalent, molecular oxygen, and copper.

Effect of phospholipid composition on the surface potential of liposomes and the activity of enzymes incorporated.

1. Microsomes of rat liver and brain and mitochondria of rat liver and guinea-pig brown adipose tissue were solubilized with the nonionic detergent Lubrol-WX and the solubilized material was incorporated into liposomes of various phospholipid composition. In proteoliposomes thus formed the kinetics of arylsulphatase, glycerol-3-phosphate dehydrogenase, monoamine oxidase and acetylcholinesterase were measured. 2. It was shown that the apparent Km values of arylsulphatase and glycerol-3-phosphate dehydrogenase were higher in liposomes prepared with negatively charged phospholipids and lower in liposomes containing positively charged organic amines, as compared with th Km value of enzymes incorporated into liposomes prepared from phosphatidylcholine alone. The opposite was true for monoamine oxidase and acetylcholinesterase, i.e. enzymes possessing cationic substrates. Phospholipid composition did not essentially influence the activity of the enzymes extrapolated for infinite substrate concentration (V values). 3. As compared with proteoliposomes made from phosphatidylcholine, the binding constant (Ka) of 8-anilino-1-naphthalene sulphonate was higher when the vesicles contained acidic phospholipids or bis(hexadecanyl)phosphate and lower when they contained organic amines. 4. A correlation between changes of the surface potential calculated from Ka values of anilino-naphthalene sulphonate and variations in apparent Km values of the four enzymes under investigation indicates that the activity of membrane-bound enzymes may be modulated by charged phospholipids due to decreasing or increasing substrate concentration in the unstirred layer, as predicted from the Boltzmann distribution.

Mitochondrial biogenesis during fungal spore germination: Purification, properties and biosynthesis of cytochrome c oxidase from Botryodiplodia theobromae

1. 1. Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from the mycelial fungus Botryodiplodia theobromae by sodium cholate and ammonium sulfate solubilization, ammonium sulfate fractionation, and DEAE-cellulose chromatography. The purified enzyme contained 6–7 nmol heme a/mg of protein. The specific activity of the purified enzyme was 2.1–2.3 · 10 3 k (min −1) per mg of protein with 15 μmol ferrocytochrome c and at pH 5.9 and optimal phosphate and Tween 80 concentrations (65 mM and 0.1%, respectively). The K m for ferrocytochrome c was determined to be 1.2–1.3 · 10 −5 M, while at infinite substrate concentration the enzyme catalyzed the oxidation of about 60 μmol of ferrocytochrome c/min per mg of protein. Sedimentation behavior and kinetic evidence suggest that the purified enzyme exists as aggregates of the single molecule. The purified B. theobromae cytochrome c oxidase with its 428 nm Soret absorption maximum may be similar if not identical to oxygenated forms of the enzyme from other fungal species. 2. 2. The purified enzyme was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to consist of seven polypeptides with the following respective molecular weights: I, 41 000; II, 28 000; III, 19 000; IV, 14 800; V, 12 800; VI, 11 500; and VII, 9300. Biosynthesis studies showed that the three highest molecular weight polypeptides of the enzyme were synthesized on mitochondrial ribosomes, and the four smaller polypeptides were products of cytoplasmic ribosomes.

Studies on the variation in Factor X activity in patients given warfarin according to the assay methods

Measurements of Factor X clotting activity by using tissue thromboplastin (T. T.) and Russell's viper venom (R. V. V.) showed the correlated result for normal rabbit plasma and human plasmas from healthy persons and patients with liver disease. However the clotting activity of Factor X in plasmas from the rabbits and patients given warfarin become variable according to the assay methods, the activity assayed by T. T. was decreased, whereas it was not so by using R. V. V.Plasma samples are tested in a series of dilutions (D) and the coagulation time (t) measured with assay system using T. T., R. V. V. and hepaplastin test (H. T.).When t is plotted against D, a straight line was obtained. Extrapolation to infinite substrate concentration gives the minima coagulation time (tmin). Normal or liver disease plasma display the same value for tmin, when using any assay systems. Plasma from persons or rabbits given warfarin display the same value for tmin when using R. V. V. or H. T., but not by T. T.These results show that clotting activity of Factor X assayed by T. T. is disturbed with competitive inhibitors probably Protein Induced by Vitamin K Absence or Antagonists (PIVKA) but assayed by R. V. V. or H. T. is not inhibited, as suggested by HEMKER.The value of amidolytic activity of Factor X is higher than that of its clotting activity assayed by R. V. V. for plasma from the persons or rabbits given warfarin.It is likely that there might be some substances which have no clotting activity but amidolytic activity associated with Factor X.Further studies might be necessary to clarify the relationship between the clotting activity and the amidolytic activity of Factor X.

Surface change of biological membranes as a possible regulator of membrane-bound enzymes.

Glycerol‐3‐phosphate dehydrogenase of insect thoracic muscle mitochondria and arylsulphatase C of rat liver microsomes were inhibited by anionic surface‐active agents oleate, palmitoyl‐CoA and sodium dodecylsulphate, and activated by cationic surfactants cetyltrimethylammonium bromide and cetylpyridinium chloride. Dimethylaniline oxidase of rat liver microsomes were inhibited by cationic surfactants and activated by anionic surfactants. The inhibition of NADH dehydrogenase of rat liver submitochondrial particles by NAD+ was potentiated by the anionic surfactants and partly released by cetyltrimethylammonium bromide. These surface‐active activators and inhibitors altered apparent Km values of the enzymes but did not change the activities at infinite substrate concentration (V values). This effect of surfactants disappeared after solubilization of the membranes and re‐appeared after incorporating the solubilized enzyme into phospholipid vesicles. Cationic surfactants decreased whereas anionic surfactants increased the negative surface charge and surface potential of mitochondria, submitochondrial particles and microsomes, as measured by free electrophoresis of the particles and binding of 8‐anilino‐1‐naphthalene sulphonate. Calculations based on Km change and the change of surface potential agree well with the assumption that a change of the surface charge density alters local concentrations of the substrates and/or the products in the vicinity of the membrane. Possible role of factors affecting the surface charge of biological membranes in controlling the activity of membrane‐bound enzymes is discussed.

The determination of positive and negative co-operativity with allosteric enzymes and the interpretation of sigmoid curves and non-linear double reciprocal plots for the MWC and KNF models

(i) It is proved that only four independent constants can ever be obtained by extrapolation procedures applied to non-hyperbolic steady-state or binding data, (ii) Analysis of the algebraic graphs y x , (1/y) (1/x) , y ( y x ) and ( x y )/x is shown to require a knowledge of the sign of six curve shape determinants. In each case, the sign is a necessary and sufficient condition for a specific curve shape feature, (iii) The precise graphical effect of positive and negative co-operativity then requires the definition of two reference curves, the osculating hyperbola at zero substrate concentration, OH(0), and the osculating hyperbola at infinite substrate concentration OH(∞). These are better first order approximations than the Hill equation, (iv) Rules for determining unambiguously the sign of initial, final and overall co-operativity coefficients by inspection of non-hyperbolic binding curves are then possible, (v) These rules require that saturation data for: y= ∑ i=1 n a ix i ∑ i=0 n β ix i be fitted by computer for low concentrations to the hyperbola: OH(o)= ( -a 1 2 ψ 11 20 )x [( -a 1β 0 ψ 11 20 )+x] while regression of high substrate concentration data is to: OH(∞)= ( a n β n )x [( φ n,n-1 a nβ n )+x] . Comparisons of the best fit pseudo-kinetic constants then gives the type of co-operativity present in an unambiguous way with no assumptions as to molecular mechanism, (vi) These rules are then applied to the MWC and KNF allosteric models of ligand binding and the constraints necessary for specific curve shape effects are given, (vii) The graphical expression of positive or negative final co-operativity depends only on events at high substrate concentration but overall and initial co-operativities produce specific geometric effects depending upon the difference between behaviour of saturation data at both extremes of concentration, (viii) This apparent anomaly is explained by a discussion of the relationships between the osculating hyperbolae, the theoretical parent hyperbola and the Hill plot asymptotes.

Further investigations of the transient kinetics of alcohol oxidation catalysed by horse liver alcohol dehydrogenase

Due to the controversy over the half-of-the-sites reactivity of horse liver alcohol dehydrogenase during benzyl alcohol oxidation, we have re-investigated the transient kinetics, stoichiometry and rate parameters over a wide range of substrate concentrations (0.05 m m to 40 m m) at pH 7.0 and 8.5 and using newly determined extinction coefficients. Data were elaborated by computer analysis in order to separate the initial rapid step (burst) from the whole time-course of the reaction. It has been found that: (1) the dependence of the burst amplitude upon benzyl alcohol concentration is distinctly biphasic. In the range from 0.05 m m up to approximately 1 m m the burst amplitude is rather insensitive to changes in alcohol concentration and corresponds to 50% of the active sites of the enzyme; for alcohol concentrations greater than 1 m m this amplitude increases and reaches a value of approximately 90% when benzyl alcohol is 40 m m. (2) The steady-state initial rate is also biphasic with respect to alcohol concentration, indicative of substrate inhibition, which begins in the concentration range at which deviation from the half-burst also appears. In other words, burst amplitudes larger than 50% are concomitant with inhibition of the rate of enzyme turnover. (3) In the presence of isobutyramide the burst is larger than 50% for the whole range of concentration of the substrate and extrapolates at infinite substrate concentration to approximately 90% of the enzyme sites. (4) With deuteroethanol as substrate, the burst is larger than 50%, with or without isobutyramide, and extrapolates to approximately 95% of the enzyme sites at infinite substrate concentration. These data explain the discrepancy of results in the literature concerning the transient kinetics of alcohol oxidation. Mechanistic implications of the results (particularly the deviation from the halfof-the-sites behaviour of benzyl alcohol under inhibition conditions) are discussed.

Detection of inhibitors in reduced nicotinamide adenine dinucleotide by kinetic methods.

Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of reduced nicotinamide adenine dinucleotide. They are (a) comparison of values by kinetic methods with those measured for highly purified NADH and (b) examination of Lineweaver-Burk plots. Chromatographic inhomogeneities are correlated with deviant values for the kinetic constants of NADH preparations. Lineweaver-Burk plots that curve upward at the high concentrations or have a larger or smaller than normal slope may indicate the presence of inhibitor. As determined in bicarbonate buffer (0.11 mol/liter, pH 7.9) by use of 0.600 mmol/liter pyruvate and NADH freshly separated from impurities by chromatography on diethyl-aminoethyl-cellulose, the Km (apparent) of NADH at 25 degrees C has the value 8.11 +/- 0.71 mumol/liter (SD, n = 28) with LDH-1 (pig heart, 2.48 +/- 0.05 U per milliliter of reaction mixture, or 41.3 +/- 0.8 nmol/liter per second). Under similar conditions, the Km (apparent) of NADH has the value of 8.57 +/- 1.58 mol/liter (SD, n = 21) with LDH-5 (pig muscle, 1.77 +/- 0.03 U/ml of reaction mixture), or 29.4 +/- 0.6 nmol/liter per second). At infinite substrate concentrations with the same pH, buffer, and temperature, the Km (apparent) for NADH was 26.0 +/- 0.63 mumol/liter with LDH-1 and 23.2 +/- 4.6 mumol/liter with LDH-5.

Studies on the intramolecular and intermolecular kinetic isotope effects in pyruvate carboxylase catalysis.

A deuterium kinetic isotope effect of 2.1 was observed when [2H3]pyruvate was used as the substrate for pyruvate carboxylase. The effect is on Vmax/Km alone and disappears at infinite substrate concentration. This is interpreted to mean that the slowest step in the overall catalysis is in the half-reaction involving the carboxylation of enzymebiotin by ATP and HCO3-. A tritium intramolecular isotope effect of 4.8 and an intermolecular effect of 1.2 were also observed. The former was interpreted as the isotope effect on the "effective kcat", while the latter the one on V max/Km. With these data, the rate constant for binding of pyruvate was estimated to be 4.5 X 10(6) M-1 min-1, and the deuterium kinetic isotope effect on the catalytic step to be 3.1. Relative values for various rate constants were also obtained. Fluoropyruvate was also shown to be a substrate, reacting six times slower. A deuterium kinetic isotope effect of 1.5 was observed, which remained even at infinite substrate concentration. This is interpreted to mean that the slowest step in the overall catalysis is now the carboxylation of fluoropyruvate.

Studies on the Elimination Reaction of d-Amino Acid Oxidase with α-Amino-β-chlorobutyrate: FURTHER EVIDENCE FOR ABSTRACTION OF SUBSTRATE α-HYDROGEN AS A PROTON

Abstract d-Amino acid oxidase catalyzes the nonoxidative conversion of α-amino-β-chlorobutyrate to α-ketobutyrate, but does not catalyze its oxidation to β-chloroketobutyrate. The elimination reaction requires the oxidized flavoprotein, i.e. it does not occur with the apoenzyme or when the enzyme-bound flavin is reduced. The occurrence of this elimination reaction supports the proposal that removal of the substrate α proton is an early step in reactions catalyzed by d-amino acid oxidase. Addition of α-amino-β-chlorobutyrate to d-amino acid oxidase leads to the sequential formation of three spectroscopically recognizable complexes (I, II, and III). All of these complexes have the spectra of oxidized flavin and in addition have long wave length absorbance extending to 800 nm. They are formed with the following rate constants: ki, 2000 min-1; kii, 40; and kiii, ∼1. (ki is dependent on substrate concentration. The value given is the limiting value at infinite substrate concentration.) When an α deuterated substrate is used, ki shows a deuterium isotope effect of about 5. Therefore, formation of Complex I must involve breaking of the substrate α C—H bond. Complex I could be a complex between flavin and a substrate-derived carbanion, or, if the elimination of HCl is complete at this stage, a complex between substrate-derived enamine and flavin. Formation of Complex I cannot be rate-determining, since the turnover number for the catalytic process is 6 to 9.5 min-1. From experiments carried out with α-amino-β-chloro[α-2H]butyrate and in 2H2O, it is concluded that the action of d-amino acid oxidase on the substrate results in the formation of an enzyme-bound enamine which then undergoes two competing reactions: (a) It is released to the medium and spontaneously ketonizes to form ketobutyrate. (b) The β position of the enamine is protonated on the enzyme to form α-iminobutyric acid, which is then released and hydrolyzes to α-ketobutyrate. The proton which is added to the β position is originally derived from the α position of the substrate. The enzyme thus has the capability of sequestering the α proton of the substrate so that it does not exchange with the solvent. The relative extent to which these two processes (enamine reprotonation versus release) occur is temperature-dependent.

Phosphoenolpyruvate Carboxylase of Escherichia coli: PURIFICATION AND SOME PROPERTIES

Abstract Phosphoenolpyruvate carboxylase was purified from Escherichia coli, strain B (ATCC 11303), and its properties studied. Its amino acid composition and some of its structural properties closely resemble those of the enzyme recently purified by Smith ((1971) J. Biol. Chem. 246, 4234–4241). Steady state kinetics, studied in the presence of the activators acetyl coenzyme A, fructose diphosphate, guanosine triphosphate, and 2-mercaptoethanol, and the inhibitor, l-aspartate, suggest an allosteric enzyme going through ligand-dependent transitions between two discrete states. This conclusion is supported by measurements of optical rotation which show two extreme values attainable in saturating activator, and inhibitor, respectively. The active (R) state is characterized by a single value for Vmax and by a Km of about 0.6 mm for phosphoenolpyruvate, approached at infinite activator concentration. The alternate (T) state, stabilized by aspartate, is catalytically completely inert. The property which distinguishes this enzyme from other phosphoenolpyruvate carboxylases is that upon addition of substrate, steady state velocity develops after a lag period of variable duration. Substrate and activators shorten, but the inhibitor prolongs this lag period. Analysis of the dependence of the lag period on enzyme concentration suggests that at assay dilutions (7 µg per ml) the enzyme is present as an inactive dimer of two peptide chains (mol wt 100,000) which upon addition of substrate, or activators, associates to the active tetramer. The lag period does not completely vanish even at infinite enzyme, substrate, or activator concentrations. The residual lag period is associated with a modification of the enzyme by the substrate, for once the enzyme has reacted with substrate the residual lag period vanishes for several hours, and then gradually returns. A model, compatible with these observations, is suggested.

Liver microsomal inorganic pyrophosphate-glucose phosphotransferase and glucose-6-phosphatase. Effects of diabetes and insulin administration on kinetic parameters

1. 1. Alterations in kinetic properties of rat hepatic glucose-6-phosphatase (( D-Gluc-6-P phosphohydrolase, EC 3.1.3.9) and associated PP i-glucose phosphotransferase resulting from alloxan diabetes and insulin treatment of such diabetic animals have been studied. 2. 2. Maximal reaction velocity values extrapolated to infinite substrate concentrations ( υ max ) and Michaelis constant ( K m ) values for all substrates were assayed with liver homogenates prepared from eight untreated normal, seven alloxan-diabetic, and seven insulin-treated diabetic animals. Average enzymic activity levels (units/mg liver protein) for the various groups of animals were calculated on the basis of υ max values. 3. 3. All assays were carried out both in the absence of added detergent and with homogenates which had been supplemented with sodium deoxycholate to 0.20% (w/v). 4. 4. As determined either in the presence or absence of detergent, K m values for glucose were not affected significantly by hormonal manipulations. 5. 5. K m values of both Gluc-6-P and PP i increased significantly in diabetes, based on assays carried out in the absence of detergent. These elevations were completely reversed by administration of insulin to diabetic animals, and were not apparent when homogenates prepared from livers of all three groups of animals were supplemented with deoxycholate prior to assay. 6. 6. Most significantly, preliminary treatment of homogenates with the detergent markedly potentiated responses of activity levels, based on extrapolated υ max values, to diabetes. The ratio of Gluc-6-P phosphohydrolase activity level in diabetic animals to activity level in normal animals increased from a value of 1.5, noted in the absence of detergent, to 3.5 based on assays with deoxycholate-supplemented homogenates. Even more dramatic potentiating effects of detergents on the diabetes response were noted with PP i-glucose phosphotransferase. With this activity, the ratio of υ max based activity levels in diabetic animals to ν max -based activity levels in normal animals increased from a statistically insignificant value of 0.90 in the absence of detergent to 4.2 when assessed in the presence of deoxycholate. Administration of insulin to diabetic rats reversed this response of ν max -based activity level values. These observations confirm our previous findings on the potentiating effect of detergent on responses of enzymic activity levels to experimental diabetes, and further, reveal that this effect persists even with substrate levels extrapolated to infinity and hence is not explainable simply on the basis of alterations in affinity of enzyme for substrates under the various experimental conditions. 7. 7. These patterns of effects of diabetes on kinetic parameters of the enzyme, as assessed both in the absence and presence of detergent supplementation of homogenates, closely resemble those previously noted in acutely fasted animals and contrast sharply with glucocorticoid-induced alterations.