Linear Double-reciprocal Plots Research Articles

Characterization of the N-demethylation reactions catalyzed by horseradish peroxidase.

The hydroperoxide-supported N-demethylation reactions catalyzed by horseradish peroxidase have been characterized in detail. The ethyl hydroperoxide-supported N-demethylation of N,N-dimethylaniline by horseradish peroxidase resulted in the formation of equimolar amounts of N-methylaniline and formaldehyde with no other products detectable by high performance liquid chromatography analysis of the reaction mixture. One molecule of ethyl hydroperoxide was consumed for each molecule of formaldehyde formed in the reaction. Similar results were obtained for the hydrogen peroxide-supported N-demethylation of N,N-dimethylaniline. The horseradish peroxidase-catalyzed N-demethylation reaction could be supported by a variety of hydroperoxides, peroxides, and peracids. The turnover number for the hydrogen peroxide-supported demethylation reaction (7061) was larger than that for the ethyl hydroperoxide-supported reaction (5382) or for chloroperoxidase- or cytochrome P-450-catalyzed dealkylations. The demethylation reaction exhibited normal Michaelis-Menten saturation kinetics with respect to N,N-dimethylaniline (Km = 0.34 mM) and ethyl hydroperoxide (Km = 0.020 mM), as well as hydrogen peroxide (Km = 0.016 mM). The horseradish peroxidase-catalyzed N-demethylation reaction was not significantly inhibited by reagents which react with the superoxide anion, the hydroxyl radical, or singlet oxygen, suggesting that these activated oxygen species are not free intermediates in the reaction. There was no significant inhibition of the reaction by alpha-phenyl-t-butylnitrone, 5,5-dimethylpyrroline-N-oxide, or other free radical trapping agents. Substitution of D2O for H2O resulted in an inhibition of the reaction with a solvent isotope effect (VH2O/VD2O) of 1.6. Horseradish peroxidase did not catalyze the demethylation of N,N-dimethylaniline-N-oxide, indicating that the reaction does not proceed via N-oxidation of the amine. When the concentrations of both N,N-dimethylaniline and ethyl hydroperoxide were varied in a constant ratio a linear double reciprocal plot was obtained, which is consistent with a ping-pong kinetic mechanism for the horseradish peroxidase-catalyzed demethylation reaction.

Pancreatic porcine phospholipase A 2 catalyzed hydrolysis of phosphatidylcholine in lecithin-bile salt mixed micelles: Kinetic studies in a lecithin-sodium cholate system

Pancreatic porcine phospholipase A 2 catalyzed hydrolysis of phosphatidylcholine in bile salt lecithin mixed micelles has been studied, utilizing a series of assay mixtures for which the micellar size, weight, and composition had been experimentally determined. Under these conditions the enzymatic hydrolysis is dependent on the phosphatidylcholine-to-sodium cholate molar ratio within the mixed micelle rather than the bulk concentration of the phospholipid in the mixture: at 5 m m phosphatidylcholine, variation of the N PC N NaCH ratio from 0.2 to 2.0 increases the enzymatic activity from 82 to 933 μmol/min/mg protein. The initial rates are linear throughout the entire series of assay mixtures, the activity vs micellar concentration curves exhibit saturation behavior, and treatment of the data according to the “surface-as-cofactor” theory provides linear double-reciprocal plots which intersect in one point. The assay system should be applicable for detailed kinetic studies of lipolytic enzymes, including mammalian phospholipases which exhibit rather low activities toward lecithin-Triton X-100 mixed micelles. The system should also provide a convenient basis for mechanistic studies involving the use of inhibitory phospholipid substrate analogs.

Properties of the cyclic GMP phosphodiesterase from rat lung: Inhibition by unsaturated fatty acids

Catalytic and regulatory properties of the major form of cyclic GMP phophodiesterase (3′:5′-cyclic-GMP 5′-nucleotidohydrolase, EC 3.1.4.35) from rat lung were studied. The enzyme partially purified by a DEAE-Sepharose chromatography displayed a much higher affinity toward cyclic GMP than toward cyclic AMP, the apparent K m values being 5.7 μM and 482 μM for the guanylic and the adenylic cyclic nucleotide, respectively. In contrast, the V value for cyclic AMP was about 3-times higher than the V value for cyclic GMP. Linear double reciprocal plots of initial velocity were observed with each cyclic nucleotide. From 10 −8 to 3.3·10 −6 M, cyclic GMP did not change the hydrolysis of 1 or 10 μM cyclic [ 3H]AMP, while it became inhibitory at higher concentrations. In contrast with a calmodulin-sensitive phosphodiesterase prepared from rat brain, the lung enzyme was not stimulated by a heat-stable Ca 2+-dependent factor from rat lung or by rat brain calmodulin or by lipids including fatty acids and lysophosphatidylcholine. Various unsaturated 18- and 20-carbon fatty acids inhibited at varying degrees the cyclic GMP phosphodiesterase from rat lung. The inhibitory potency increased with the number of double bonds in the hydrocarbon chain. In contrast, the methyl esters of the unsaturated fatty acids and the saturated fatty acids of variable hydrocarbon chain lengths had no appreciable effects. A linear Hill plot of phosphodiesterase inhibition with a slope of unity was obtained with arachidonic acid up to 30 μM, suggesting only one type of inhibitory site. In this range of concentrations the inhibition was entirely reversible. Kinetics analysis demonstrated that up to 30 μM arachidonic acid was a purely competitive inhibitor with an apparent K i of 20 μM. Over 30 μM, the Hill coefficient increased progressively, indicating the binding to other inhibitory sites, while the reversibility disappared.

The effect of anions on the reaction catalyzed by lupine-nodule glutamate dehydrogenase

The kinetics of the reductive amination reaction of lupine-nodule glutamate dehydrogenase ( l-glutamate:NAD oxidoreductase (deaminating), EC 1.4.1.2) were found to vary with the identity of the ammonium salt which was used as a substrate. Normal Michaelis-Menten kinetics were obtained with (NH 4) 2SO 4 but when NH 4Cl or NH 4-acetate was varied apparent substrate inhibition was observed. Linear double-reciprocal plots were obtained with NH 4Cl and NH 4-acetate, however, if the concentration of Cl − or acetate was maintained constant by adding KCl or K-acetate. Chloride and acetate were subsequently found to cause linear noncompetitive inhibition with respect to NH 4 + and the apparent substrate inhibition by NH 4Cl and NH 4-acetate can be explained as the result varying a substrate and a noncompetitive inhibitor in constant ratio. Other anions were also found to be inhibitors of the glutamate dehydrogenase reaction; I − caused parabolic noncompetitive inhibition with respect to NH 4 + and NO 3 − caused slope-parabolic noncompetitive inhibition with respect to all three substrates of the reductive amination reaction. For the oxidation deamination reaction, Cl − was a linear competitive inhibitor with respect to both NAD and l-glutamate whereas NO 3 − caused parabolic competitive inhibition with respect to these reactants. To explain the results, it is proposed that anions bind to an allosteric site and cause a change in some of the rate constants of the reaction. Specifically, the results are consistent with anions causing decreases in the rates of association of NADH and 2-oxoglutarate with the enzyme and an increase in the rate of dissociation of NAD.

Malic enzyme of Chromatium vinosum

Malic enzyme of the phototropic bacterium Chromatium vinosum strain D that lacks malate dehydrogenase was partially purified yielding a specific activity of 55 units/mg protein. The constitutive enzyme with a molecular weight of 110,000 and a pH optimum of 8.0 was absolutely dependent on the presence of a monovalent cation (NH4+, K+, Cs+, or Rb+) as well as a divalent cation (Mn2+, or Mg2+). The enzyme was inhibited by oxaloacetate, glyoxyate, and NADPH. The K0.5 value for L-malate and the inhibition constants for oxaloacetate and glyoxylate are dependent on the concentration of the monovalent cation, whereas the Km value for NADP (18 microM) and the KI value for NADPH (42 microM) are independent. Throughout all kinetic measurements hyperbolic saturation curves and linear double reciprocal plots were obtained.

Deviations from Michaelis-Menten kinetics. The possibility of complicated curves for simple kinetic schemes and the computer fitting of experimental data for acetylcholinesterase, acid phosphatase, adenosine deaminase, arylsulphatase, benzylamine oxidase, chymotrypsin, fumarase, galactose dehydrogenase, beta-galactosidase, lactate dehydrogenase, peroxidase and xanthine oxidase.

The possible graph shapes for one-site/two-state and substrate-modifier models are discussed. The two-state model is a version of the Monod-Wyman-Changeux model and gives a rate equation with 240 denominator terms. Discussion in terms of K and V effects is not possible. A simplified version of the mechanism can be shown to give v-versus-[S] curves that are either sigmoid or non-sigmoid. They may show substrate inhibition or no final maximum, and the double-reciprocal plots can be concave up or down. The corresponding binding model is determined by only two constants and gives a linear double-reciprocal plot. The substrate-modifier mechanism is a simple example of a mechanism where inclusion of catalytic steps leads to a genuine increase in degree of the rate equation. The v-versus-[S] curve can show such complexities as two maxima and a minimum, and the double-reciprocal plot can cross its asymptote twice, proving the rate equation to be 4:4. A simplified version is 3:3, and analysis shows that at least 18 of the 27 double-reciprocal plots that can arise with 3:3 functions are possible with this particular mechanism. Representative double-reciprocal and Scatchard plots are presented for several sets of rate-constant values. It is concluded that relatively simple mechanisms give pseudo-steady-state rate equations of high degree and considerable complexity. With extended ranges of substrate concentrations there is every reason to believe that experimental data would show the sort of deviations from Michaelis-Menten kinetics seen with calculated curves for such simple mechanisms. Narrow ranges of substrate concentration, on the other hand, would lead to inflexions and curvature being overlooked. It is not possible to discuss such deviations from Michaelis-Menten kinetics in terms of kinetic constants such as Km and V, and, in general, it is also difficult to see any simple way to explain intuitively such features as sigmoidicity, substrate inhibition, double-reciprocal convexity and decrease in degree by cancellation of common factors between numerator and denominator of rate equations. These conclusions apply with even more force when catalytic steps are included, for then the rate equations, are for multi-site mechanisms, of higher degree, allowing increasingly complex curve shapes. A number of enzymes were studied and initial-rate data were fitted by computer.(ABSTRACT TRUNCATED AT 400 WORDS)

Saturable high affinity binding of low density and high density lipoprotein by parenchymal and non-parenchymal cells from rat liver

Freshly isolated parenchymal liver cells bind both low density lipoprotein (LDL) and high density lipoprotein (HDL). With increasing concentrations of LDL and HDL the amount of cell-associated radioactivity approaches saturation and a linear double-reciprocal plot for the binding is obtained. The binding of LDL and HDL to isolated non-parenchymal liver cells is also saturable and the maximal binding of LDL and HDL per mg cell protein is 4–5 times higher than with parenchymal cells. It is suggested that the presence of a 4–5 fold higher concentration of lipoprotein receptor (for LDL and HDL) on non-parenchymal cells as compared to parenchymal cells explains the 4–5 times higher uptake of lipoproteins by the non-parenchymal liver cells, observed in vivo .

Pathways of cycloleucine transport in killifish small intestine.

By using blocking concentrations of competitors, i.e., concentrations of other neutral amino acids that cause maximal inhibition, cycloleucine transport into slices or everted sacs of killifish (fundulus heteroclitus) small intestine could be partitioned into three pathways. One is apparently not mediated, a second is inhibited by all neutral amino acids tested (component 1), and a third, is inhibited by alpha-aminocarboxylic acids (component 2), but not by beta-alanine or taurine. Both mediated pathways were Na dependent, and each yielded a linear double reciprocal plot of initial slice uptake vs. cycloleucine concentration. Apparent Kt and Vmax values for component 1 were 0.03 mM and 33 pmol/mg tissue per 3 min, respectively; corresponding values for component 2 were 0.12 mM and 28 pmol/mg tissue per 3 min. Additional experiments with an intestinal brush border membrane vesicle preparation indicate that these mediated components reflect true differences in carrier specificity rather than the differential effects of inhibitors on metabolism or on the Na gradient that drives cycloleucine transport.

Multiple forms of cyclic nucleotide phosphodiesterase from bovine carotid artery smooth muscle

DEAE-cellulose chromatography, in the presence and absence of Ca 2+, of the 16,000 g supernatant from bovine carotid artery smooth muscle has been used to separate four different types of cyclic nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) activity, designated types A, B, C, and D. Type A is a high affinity, cyclic AMP-specific form of phosphodiesterase ( K m = 1.6 μM) and elutes at relatively high ionic strength. Type B is a high affinity ( K m = 2 μM), cyclic GMP-specific form which elutes at low ionic strength. Type C is a mixed substrate form, displaying anomalous kinetics for the hydrolysis of both cyclic AMP and cyclic GMP. It elutes from DEAE-cellulose at an ionic strength intermediate to that of types A and B. Type D is also a mixed substrate form of phosphodiesterase. However, its elution pattern from DEAE-cellulose differs, depending on whether Ca 2+ is present or not, suggesting a Ca 2+-dependent interaction between this enzyme form and the acidic Ca 2+-dependent regulator protein (CDR). The hydrolytic activity of type D is stimulated by CDR, and activation requires the simultaneous presence of Ca 2+ and CDR. Kinetic analysis of cyclic AMP hydrolysis by type D gives a linear double reciprocal plot; activation has no effect on the K m but increases the velocity approximately sixfold. Activation of cyclic GMP hydrolysis apparently affects both the K m and V. At all concentrations tested, the degree of activation is higher with cyclic AMP than with cyclic GMP. It is suggested that while the activable form of phosphodiesterase may play a relatively minor role in the overall hydrolysis of cyclic nucleotides, Ca 2+-dependent activation may have a more important role in regulating the level of cyclic AMP than that of cyclic GMP in vascular smooth muscle.

Electron-transferring enzymes in the plasma membrane of the Ehrlich ascites tumor cell

The plasma membrane of the Ehrlich ascites tumor cell contains an NADH dehydrogenase. This activity was shown not to be due to contamination by other subcellular membranes. A variety of electron acceptors have been compared as to rate with the following result: ferricyanide greater than cytochrome c greater than cytochrome b5 greater than glyoxylate greater than dichlorophenolindophenol. Oxygen acceptance could not be detected. The optimum assay temperature and pH ranges were 30--40 degrees C and pH 6--8, respectively. With respect to either NADH or ferricyanide, the kinetics yielded linear double-reciprocal plots. Inhibition of the enzyme by sulfhydryl reagents could be blocked by excess NADH. Detergents such as Triton X-100 or cholate resulted in solubilization of the enzymatic activity, but phospholipase A2 did not. The activity differed from that of the mitochondria in that it was not inhibited by rotenone or antimycin A. The possible involvement of NADH oxidation in the energetics of plasma membrane transport is discussed.

Biosynthesis of fatty alcohols, alkane-1,2-diols and wax esters in particulate preparations from the uropygial glands of white-crowned sparrows ( Zonotrichia leucophrys)

Biosynthesis of sebaceous gland waxes was studied with the uropygial gland of the white-crowned sparrow as the experimental tissue. A 27,000 g particulate preparation from this gland catalyzed reduction of palmitoyl-CoA to hexadecanol at an optimum pH near 5.0 with NADPH as the preferred reductant. At low protein concentrations, palmitoyl-CoA inhibited the reductase and bovine serum albumin prevented this inhibition. An apparent K m of 0.3 m m was calculated for palmitoyl-CoA from linear double-reciprocal plots ignoring the inhibitory concentration of the substrate. An apparent K m of 3 m m was calculated for NADPH from linear double-reciprocal plots. Palmitoyl-CoA reduction was inhibited by thiol directed reagents such as p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide. The particulate fraction also catalyzed esterification of hexadecanol with endogenous C 16 and C 18 acyl moieties with an optimum pH of 7.5. Stimulation of esterification of hexadecanol by ATP and CoA as well as by low concentrations of palmitoyl-CoA suggests that the CoA esters of fatty acids are involved in esterification. Tween-20 stimulated esterification of hexadecanol and hexadecyl dodecanoate was the major wax ester formed in the presence of Tween-20 suggesting that the C 12 acid of Tween-20 participated in esterification. Ignoring the inhibitory concentrations of hexadecanol (>0.2 m m), an apparent K m of 0.1 m m was calculated from linear double-reciprocal plots. α-Hydroxylation of palmitic acid was demonstrated in cell-free extracts of the uropygial gland. A 27,000 g particulate preparation from the gland catalyzed the reduction of α-hydroxypalmitic acid to hexadecane-1,2-diol with NADPH as the preferred reductant at an optimum pH near 6.5. This reduction required both ATP and CoA, suggesting that α-hydroxyacyl-CoA was the true substrate for the reductase. With stereospecifically labeled NADP 3H, it was shown that both acyl-CoA reduction and α-hydroxy acid reduction involved transfer of the hydride specifically from the B-side of the nicotinamide ring of NADPH. Subcellular fractionation using sucrose density gradient centrifugation strongly suggested that the enzymes which catalyzed reduction of palmitoyl-CoA and α-hydroxypalmitic acid as well as the esterification of hexadecanol are localized in the microsomal membranes of the gland.

Biosynthesis of alkane-2,3-diols: Enzymatic reduction of 3-hydroxyoctadecane-2-one to octadecane-2,3-diol by a NADPH (B-Side) specific microsomal reductase from the uropygial glands of ring-necked pheasants ( Phasianus colchicus)

Cell-free preparations from the uropygial gland of ring-necked pheasant catalyzed the reduction of a synthetic R,S-mixture of 3-hydroxyl[3- 14C]octadecane-2-one (acyloin) to a mixture of threo- and erythro-[3- 14C]octadecane-2,3-diol, the final step in the postulated pathway for the biosynthesis of alkane-2,3-diols. The product of enzymatic reduction was identified by Chromatographic techniques and chemical degradation studies. The acyloin reductase showed a pH optimum near 4.0 and specificity for NADPH. With stereospecifically labeled [ 3H]NADPH, it was shown that acyloin reductase preferentially transferred hydride from the B-side of the nicotinamide ring to the acyloin. A typical Michaelis-Menten substrate saturation was observed for the acyloin and an apparent K m of 70 μ m was calculated from linear double reciprocal plots. Acyloin reductase was inhibited by thioldirected reagents such as p-chloromercuribenzoate and N-ethylmaleimide. Subcellular fractionation of the gland homogenates using sucrose density gradient centrifugation showed that acyloin reductase activity coincided with NADPH:cytochrome c reductase activity, strongly suggesting that acyloin reductase is localized in the microsomal membranes.

Binding, interiorization and degradation of cholesterol ester-labelled chylomicron-remnant particles by rat hepatocyte monolayers

1. The cholesteryl ester of isolated chylomicron-remnant particles was efficiently degraded by hepatocyte monolayers. The degradation was sensitive to metabolic inhibitors. 2. With increasing amounts of remnant cholesteryl ester the rate of uptake approached saturation and conformed to a linear double-reciprocal plot. The V(max.) was determined as 80ng of cholesteryl ester/h per mg of protein and the apparent K(m) as 1.4mug of cholesteryl ester per mg of protein. The time course for the uptake and hydrolysis suggested that binding of particles to the cell surface preceded the degradation. 3. Cholesteryl esters of native chylomicrons were degraded to a much smaller extent and their presence had only a small inhibitory effect on the degradation of chylomicron remnants. Intestinal very-low-density lipoproteins were degraded somewhat faster than chylomicrons, and caused more inhibition of remnant degradation. Rat high-density lipoproteins inhibited the hydrolysis of remnant cholesteryl ester by up to 50%, but had less influence on the amount of cholesteryl ester that was bound to the cells. Serum decreased both the uptake and hydrolysis, whereas d=1.21 infranatant had no effect. 4. The cholesteryl ester hydrolysis after the uptake by the cells was inhibited by chloroquine and by colchicine. Only 28-36% of the unhydrolysed cholesteryl ester could be released from these cells by trypsin treatment, indicating that the major portion was truly intracellular. The particles that could be released from the cell surface by trypsin and those remaining in the medium had the same triacylglycerol/cholesteryl ester ratio as the added remnant particles. Significant amounts of denser particles were thus not formed during contact with the cell surface. 5. The presence of heparin, as well as preincubation of the cells with heparin, increased the uptake of chylomicron remnants. This effect was most marked in the presence of serum. A much smaller proportion of the other serum lipoproteins was taken up, and this proportion was not increased by heparin.

Glycerokinase in human adipose tissue.

The presence of glycerokinase has been demonstrated in human omental and subcutaneous adipose tissue. The enzyme reaction showed a linear time course for 5 min at 30 C and pH optima at pH 7.6 and 9.0. Saturation of the enzyme was observed at 1.8 mM adenosine triphosphate (ATP) and the double reciprocal plot of activity vs. ATP concentration was nonlinear giving two apparent Km values of 0.094 and 0.518 mM. The apparent Km for glycerol, 0.112 mM, was obtained from a linear double reciprocal plot, and the enzyme was saturated at about 0.4 mM glycerol. The activity of glycerokinase in human adipose tissue excised under general anaesthesia was low and was unrelated to adipose cell size or the degree of obesity of the subject from whom the fat was obtained.

Stimulation of human platelet guanylate cyclase by fatty acids.

Guanylate cyclase from human platelets was over 90% soluble, even when assayed in the presence of Triton X-100. A time-dependent increase in activity occurred when the enzyme was incubated at 37 degrees and this spontaneous activation was prevented by dithiothreitol. Arachidonic acid stimulated the soluble enzyme activity approximately 2- to 3-fold. Linear double reciprocal plots of guanylate cyclase activation as a function of arachidonic acid concentration were obtained with a Ka value of 2.1 muM. A Hill coefficient of 0.98 was obtained indicating that one fatty acid binding site is present for each catalytic site. Concentrations of arachidonic acid in excess of 10 muM caused less than maximal stimulation. Dihomo-gamma-linolenic acid and two polyunsaturated 22 carbon fatty acids stimulated the activity of guanylate cyclase to the same degree as did arachidonic acid. The methyl ester of arachidonic acid was much less effective. Diene, monoene, and saturated fatty acids of various carbon chain lengths as well as prostaglandins E1, E2, and F2alpha, had little or no effect. These data indicate that the structural determined required for stimulation by fatty acids of soluble platelet guanylate cyclase is a 1,4,7-octatriene group with its first double bond in the omega6 position. This structural group is similar to the substrate specificity determinants of fatty acid cyclooxygenase, the first enzyme of the prostaglandin synthetase complex. However, conversion of arachidonic acid to a metabolite of the cyclooxygenase pathway did not appear to be required for activation of the cyclase since activation occurred in the 105,000 X g supernatant fraction and pretreatment of this fraction with aspirin did not alter the ability of arachidonic acid to activate guanylate cyclase. Kinetic studies showed that the stimulation of guanylate cyclase by arachidonic acid is primarily an effect on maximal velocity. Arachidonic acid did not alter the concentration of free Mn2+ required for optimal activity. It is concluded that the activity of the soluble form of guanylate cyclase in cell-free preparations of human platelets can be increased by a lipid-protein interaction involving specific polyunsaturated fatty acids.

Studies on the kinetic mechanism of oxidative phosphorylation.

The kinetics of the synthesis of ATP from ADP and Pi by beef heart submitochondrial particles were examined. When Pi was the variable substrate positive cooperativity was observed, whereas if ADP was varied, linear double reciprocal plots were obtained. The analog of Pi, thiophosphate, was a noncompetitive inhibitor of ATP synthesis with respect to ADP, while the analog of ADP, AMP (CH2)P, was an uncompetitive Pi leads to ATP exchange inhibitor. The kinetics of the initial velocity isotopic exchanges of oxidative phosphorylation were also examined. When the Pi leads to ATP exchange was examined, it was found that if ADP concentration was held constant while ATP and Pi concentrations were varied at a constant ratio, linear double reciprocal plots were obtained. However, if Pi concentration was held constant and ADP and ATP concentrations were varied at constant ratio, apparent substrate inhibition was observed. The 2, 4-dinitrophenol-sensitive ADP leads to ATP exchange showed linear double reciprocal plots regardless of which components were varied. These results are interpreted to indicate that in the direction of ATP synthesis, the reaction is ordered, with Pi adding to the enzyme before ADP addition.

Endorphin-partial purification and demonstration of multiple components

1. 1. Partially purified endorphin extracted from C57BL/6J mice competitively inhibits 3H-dihydromorphine (DHM) and 3H-naltrexone binding to C57BL/6J mouse brain homogenates. 2. 2. The inhibition of binding as a function of endorphin concentration yields a linear double reciprocal plot which extrapolates to displacement of 100% of the bound opiate. 3. 3. Gel filtration of partially purified endorphin indicates multiple components.

Purine nucleoside phosphorylase of rabbit liver. Mechanism of catalysis.

Initial velocity studies and product inhibition patterns for purine nucleoside phosphorylase from rabbit liver were examined in order to determine the predominant catalytic mechanism for the synthetic (forward) and phosphorolytic (reverse) reactions of the enzyme. Initial velocity studies in the absence of products gave intersecting or converging linear double reciprocal plots of the kinetic data for both the synthetic and phosphorolytic reactions of the enzyme. The observed kinetic pattern was consistent with a sequential mechanism, requiring that both substrates add to the enzyme before products may be released. The product inhibition patterns showed mutual competitive inhibition between guanine and guanosine as variable substrates and inhibitors. Ribose 1-phosphate and inorganic orthophosphate were also mutually competitive toward each other. Other combinations of substrates and products gave noncompetitive inhibition. Apparent inhibition constants calculated for guanine as competitive inhibitor and for ribose 1-phosphate as noncompetitive inhibitor of the enzyme, with guanosine as variable substrate, did not vary significantly with increasing concentrations of inorganic orthophosphate as fixed substrate. These results suggest that the mechanism was order and that substrates add to the enzyme in an obligatory order. Dead end inhibition studies carried out in the presence of the products guanine and ribose 1-phosphate, respectively, showed that the kinetically significant abortive ternary complexes of enzyme-guanine-inorganic orthophosphate (EQB) and enzyme-guanose-ribose 1-phosphate (EAP) are formed. The results of dead end inhibition studies are consistent with an obligatory order of substrate addition to the enzyme. The nucleoside or purine is probably the first substrate to form a binary complex with the enzyme, and with which inorganic orthophosphate or ribose 1-phosphate may interact as secondary substrates. The evidences presented in this investigation support an Ordered Theorell-Chance mechanism for the enzyme.

Acyl coenzyme A dehydrogenases and electron-transferring flavoprotein from beef heart mitochondria

Electron-transferring flavoprotein (ETF) and long-chain acyl coenzyme A (CoA) dehydrogenase (LC-AD) have been purified essentially to homogeneity from beef heart (BH) mitochondria and partially characterized. The spectra of the major yellow acyl CoA dehydrogenase from BH mitochondria, both oxidized and when bleached with C 16CoA, were found to resemble those of pig liver (PL) LC-AD. The subunit molecular weight was found to be about 38,000 both by Na-dodecyl sulfate gel electrophoresis and by minimal molecular weight based on flavin content (A 450, ϵ = 11.3 × 10 3 cm −1 m −1). The enzyme is probably a tetramer with no interchain disulfide bonds. When assayed in the presence of ETF, relative activities are C 8CoA > C 16CoA ⪢ C 4CoA. These findings show that physicochemical and specificity characteristics do not coincide in the pig liver and the beef heart enzymes. The BH ETF is similar to the PL ETF in its spectra, in subunit molecular weight determined by minimal molecular weight (based on flavin content as A 438), by Na-dodecyl-SO 4 gel electrophoresis, the absence of interchain disulfide bonds, V̄ p, and the presence of two subunits/molecule. There were some changes in the amino acid composition concomitant with a decrease in apparent isoelectric point. The pig and beef enzymes were reactive with each other. The turnover number of the beef heart system at “saturating” ETF was 100 mol of 1, 6-dichlorophenol indophenol reduced/min/ mol of LC-AD. Abnormally low activity at low ETF concentrations as compared to high ETF concentrations was seen with the beef heart enzymes as with the pig liver system previously studied and again a material obtained during purification of the ETF similar to the “factor” from pig liver (based on chromatographie and disc-gel electrophoretic behavior) stimulated the low activity, while the high-ETF activity was relatively unaffected, permitting linear double-reciprocal plots over wide ranges of ETF concentration. Fatty-acid-free bovine serum albumin (BSA-FAF) could mimic this effect at equivalent protein concentrations (50–100 μg), as could increased LC-AD concentration and, to a lesser extent, limited aging. Studies of activity at very high concentrations of C 16CoA revealed a marked high-substrate inhibition with activity peaking at about 4 μ m, the reported critical micelle concentration for C 16CoA. The addition of BSA-FAF resulted in more “normal” v vs [S] curves, and although the substrate inhibition was still present it was less severe. The K m for C 16CoA in the presence of BSA-FAF is about 1 μ m. These results suggest that the inhibitory species may be the C 16CoA micelle, and the BSA-FAF may reverse or alleviate the inhibition by binding acyl CoA in a manner analogous to its binding of fatty acid anions.

The effect of oxygen concentration on the steady-state kinetics of the solubilized cytochrome [formula omitted] oxidase

1. 1. The steady-state kinetics of ascorbate oxidation as a function of oxygen concentration was measured with a solubilized cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) preparation. 2. 2. Linear double reciprocal plots were obtained at various fixed concentrations of ascorbate, cytochrome c and cytochrome aa 3. 3. 3. The results are interpreted in terms of an oxidase model similar to that put forward by Minnaert in 1961 (Minnaert, K. (1961) Biochim. Biophys. Acta 50, 23–34). 4. 4. The K m for oxygen at infinite cytochrome c concentration is 0.95 μM and the intramolecular rate constant for the transfer of electrons from cytochrome c to cytochrome aa 3 is 400 s −1. According to the model, this implies that the second order rate constant for the reaction between oxygen and the oxidase is 9.5 × 10 7 M −1 · s −1.