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1.
Barbara Baranowska Grzegorz Terlecki Tadeusz Baranowski 《Molecular and cellular biochemistry》1984,64(1):45-50
Summary The mechanism of activation by inorganic phosphate and ATP of cardiac muscle pyruvate kinase was studied with the aid of steady-state kinetics. The enzyme was purified to homogeneity to a final specific activity of 400 units/ mg (phosphate buffer, pH 7.6, 25 °C). At pH 7.6 the enzyme displays Michaelis-Menten kinetics with respect to both its substrates, phosphoenolpyruvate and ADP. Substrate kinetic constants are: app.Km(phosphoenolpyruvate) –0.04 mM, app.Km(ADP) =0.22 mM. Under the conditions used in the standard assay the specific activity is greatly enhanced by inorganic phosphate (50 mM) or ATP (2.5 mM). Each of these modifiers, acting separately, increases the Vmax without seriously affecting Michaelis constants and Hill coefficients. In the presence of both Pi and ATP, only a decrease in Vmax was observed.The kinetics of activation by inorganic phosphate of pyruvate kinase was examined. Studying the effect of varying concentrations of Pi on the initial rate we obtained a hyperbolic saturation curve with the app. Km(Pi) = 20 mM and Vmax = 167 units/ mg. The evidence is presented that inorganic phosphate is a substrate for a side reaction catalyzed by cardiac pyruvate kinase. It is shown that in the presence of pyruvate, inorganic phosphate and ATP in the assay system, Pi is incorporated into acid-labile products of this reaction, inorganic pyrophosphate being one of them.These findings indicate the existence of an alternative reaction catalyzed by pyruvate kinase by which energy may be stored in the form of inorganic pyrophosphate.Abbreviations PEP
phosphoenolpyruvate
- Pi
inorganic phosphate
- TEA
triethanolamine
- EDTA
ethylenediaminetetraacetate 相似文献
2.
Summary About 25% of total pyruvate kinase activity in muscle appears in a bound form which is insoluble in water or diluted salt solutions at pH 5.8. That activity is associated with the ribonuc-leoprotein complexes and is soluble at high ionic strength. A procedure is described for the purification and crystallization of this enzyme form herein called pyruvate kinase MB and water soluble form MA.Crystalline nucleoproteins are composed of active and inactive RNA-protein complexes with varying RNA content. By fractional crystallization and gel filtration a number of crystalline complexes were separated, two of them highly purified. One preparation was homogenous, contained 0.5% RNA and had a specific activity of 265 U/mg protein, the other one 10% RNA and 200 U/mg protein respectively.Forms MA and MB share the same protein as shown in immunodiffusion test with the anti-MA sera. They differ in solubility and stability in diluted solutions. In Tris-HCl buffer, pH 7.6 form MB is rapidly inactivated whereas form MA is quite stable under the same conditions. Both forms have different Km for phosphoenolpyruvate and ADP and Vmax as well.Digestion of pyruvate kinase MB with RNase was without marked effect on specific activity of the enzyme.The presence of numerous ribonucleoprotein complexes with a polynucleotide content in the range of 0.5 to 20% and specific activity of 160–220 U/mg protein suggests the control by RNA binding of pyruvate kinase activity from human skeletal muscle.This work was supported by a grant from the Biochemical and Biophysical Committee of the Polish Academy of Sciences. 相似文献
3.
Pyruvate kinase from Propionibacterium shermanii was shown to be activated by glucose-6-phosphate (G-6-P) at non-saturating phosphoenol pyruvate (PEP) concentrations but other glycolytic and hexose monophosphate pathway intermediates and AMP were without effect. Half-maximal activation was obtained at 1 mM G-6-P. The presence of G-6-P decreased both the PEP0.5V and ADP0.5V values and the slope of the Hill plots for both substrates. The enzyme was strongly inhibited by ATP and inorganic phosphate (Pi) at all PEP concentrations. At non-saturating (0.5 mM) PEP, half-maximal inhibition was obtained at 1.8 mM ATP or 1.4 mM Pi. The inhibition by both Pi and ATP was largely overcome by 4 mM G-6-P. The specific activity of pyruvate kinase was considerably higher in lactate-, glucose- and glycerol-grown cultures than that of the enzyme catalysing the reverse reaction, pyruvate, phosphate dikinase. It is suggested that the activity of pyruvate kinase in vivo is determined by the balance between activators and inhibitors such that it is inhibited during gluconeogenesis while, during glycolysis, the inhibition is relieved by G-6-P.Abbreviations PEP
phosphoenolpyruvate
- G-6-P
glucose-6-phosphate
- Pi
inorganic phosphate 相似文献
4.
Summary About 25% of total pyruvate kinase activity in human skeletal muscle is associated with the ribonucleoprotein complexes soluble in salt solutions of high ionic strength. These complexes, called form MB, crystallize readily from 48% saturated ammonium sulfate at pH 5.6.Crystalline preparations represent a heterogenous population of ribonucleoprotein complexes displaying a graduated activity and a variable RNA content. Free protein was not detected in the preparations.Fractionation of crystalline complexes in salt solutions of varying ionic strength and pH, followed by gel filtration on Sephadex G-200 led to the separation of two nucleoprotein fractions with very high specific activity. Fractions containing 30% RNA and 85% RNA respectively revealed a specific activity of 660–670 U/mg protein at 25°C.Pyruvate kinase form MA was extracted from muscle homogenate with distilled water, purified to homogeneity and crystallized. It contained less than 0.2% RNA and had a specific activity of 270 U/mg. Active ribonucleoprotein complexes gave in double immunodiffusion test the precipitation bands with the anti-MA sera at the same protein concentration of both antigens, MB and MA.Pyruvate kinase MB with high activity is sensitive to treatment with RNase. Digestion with RNase for 10 min at 25°C diminished the initial specific activity to about one third. Similar residual activity was found in crystalline ribonucleo protein complexes with low RNA content (3.5–20% RNA) which are resistant to further inactivation by RNase.These results implicate the enhancement and control of pyruvate kinase activity by RNA bound to the enzyme.This work was supported by a grant from the Biochemical and Biophysical Committee of Polish Academy of Sciences. 相似文献
5.
Beate Schering Erich Eigenbrodt Dietmar Linder Wilhelm Schoner 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,717(2):337-347
(1) Pyruvate kinase type M2 from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of Mr = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M2, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K0.5 value for phosphoenolpyruvate is 0.26 mM (nH = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (nH = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The Km value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The Km value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M2 from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M2 from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M2 may represent a separate form of the pyruvate kinase type M2 family. 相似文献
6.
A single form of pyruvate kinase was isolated from the green alga Chlamydomonas reinhardtii Dang. (Chlorophyta) and partially purified over twentyfold, yielding a final specific activity of 2.68 μmol pyruvate produced-min-1.mg-1 protein. Studies of its physical characteristics reveal that the pyruvate kinase is heat stable, is partially inactivated by sulfhydryl reagent N-ethylmaleimide, and has a pH optimum at 6.8 and a native molecular mass of 224 kDa. Immunological precipitation and western blotting, using antibodies raised against Selenastrum minutum Naeg. (Chlorophyta) cytosolic pyruvate kinase, reveal that C. reinhardtii pyruvate kinase possesses a subunit molecular mass of 57 kDa, indicating a homo-tetrameric structure. This enzyme exhibits an absolute requirement for a divalent cation that can be fulfilled, by Mg2+. The monovalent cation K+ acts as a strong activator. The Km values for phosphoenolpyruvate and adenosine diphosphate (ADP) are 0.16 mM and 0.18 mM, respectively. The enzyme is capable of using other nucleotides with Vmax for UDP, GDP, IDP, and CDP of 70%, 55%, 53%, and 25% of that with ADP, respectively. Dihydroxyacetone phosphate, ribulose 1,5-bisphosphate, adenosine monophosphate (AMP), ribose-5-phosphate, and glyceraldehyde-3-phosphate are activators, whereas glutamate, orthophosphate, adenosine triphosphate (ATP), citrate, isocitrate, malate, oxalate, phosphoglycolate, and 2,3-diphosphoglycerate are potent inhibitors of this enzyme. Dihydroxyacetone phosphate can reverse the inhibition by glutamate and phosphate. These properties are discussed in light of pyruvate kinase regulation during anabolic and catabolic respiration. Substrate interaction and product inhibition studies indicate that ADP is the first substrate bound to the enzyme and pyruvate is the last product released (Ordered Bi Bi mechanism). 相似文献
7.
D-Fructose 2,6-bisphosphate: a naturally occurring activator for inorganic pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase in plants 总被引:20,自引:0,他引:20
Inorganic pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase from mung beans () was activated markedly by D-fructose 2,6-bisphosphate, with a KA of about 50 nM. The enzyme exhibited hyperbolic kinetics both in the absence and presence of the activator. D-Fructose 2,6-bisphosphate (1 μM) decreased the Km for D-fructose 6-phosphate 67-fold (from 20 mM to 0.3 mM) and increased the Vmax 15-fold; these two effects combined to give a 500-fold activation at 0.3 mM D-fructose 6-phosphate. In contrast, ATP:D-fructose 6-phosphate 1-phosphotransferase from the same source was found not to be affected by D-fructose 2,6-bisphosphate.A natural activator for inorganic pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase was isolated from mung-bean extracts and identified as D-fructose 2,6-bisphosphate. 相似文献
8.
The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent Vmax values and decreased the Km values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The Km values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed. 相似文献
9.
Ritchie J. Feuers Daniel A. Casciano 《Biochemical and biophysical research communications》1984,124(2):651-656
The addition of glucagon to hepatocytes in primary culture produced a rapid and sustained increase in the Km ( phosphoenol pyruvate) of pyruvate kinase. The low Km () form of the enzyme was seen when cells were retreated with insulin, demonstrating a short-term regulation mechanism. Injections of insulin, glucagon or glucagon followed by insulin demonstrated that a similar mechanism occurs . Results from longer times after injection indicated that another mechanism occurs when altered activity was the result of changes in Vmax and not Km. Thus, a dual mechanism for regulation of pyruvate kinase occurs. A rapid responding system functions by modification of the enzyme, while a long-term system functions by altering the rate of synthesis, thus changing the amount of enzyme present. 相似文献
10.
Maurice Chan 《Biochemical and biophysical research communications》2004,326(1):188-196
The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with Km of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a Ki of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection. 相似文献
11.
Kenneth B. Storey 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1985,155(3):339-345
Summary Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withK
m values of 0.27 and 0.04 mM, respectively.K
m for Mg2+ was 0.60 mM andK
a for K+ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentK
i values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theK
m for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P2 did not activate the enzyme but reversed inhibitions by ATP and arginine-P.Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S0.5=0.32 mM andn
H=1.43.K
m values for ADP and Mg2+ were 0.30 and 0.80 mM, respectively with aK
a for K+ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P2 with an apparentK
a of 1.5 M. Fructose-1,6-P2 at 0.1 mM reduced S0.5 for P-enolpyruvate to 0.05 mM andn
H to 1.0.Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P2 is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowK
m for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P2. 相似文献
12.
The effects of urea, cations (K+, NH4, Na+, Cs+, Li+), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle, were analyzed in two anuran amphibians, an estivating species, the spadefoot toadScaphiopus couchii, and a semi-aquatic species, the leopard frogRana pipiens. Urea, which accumulates naturally to levels of 200–300 mM during estivation in toads, had only minor effects on the Vmax, kinetic constants and pH curves of PK from either species and no effects on PFK Vmax or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the Vmax of toad PFK and of PK from both species and altered the Km values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the Vmax of PK from either species was reduced by more than 80% by the addition of 200 mM NH4Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the Km values for substrates of toad lactate dehydrogenase and strongly reduced the Vmax of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes. Rather, we found that urea is largely a non-perturbing solute for anuran enzymes (I50 values were>1 M for both PK and PFK in both species) and we propose that its accumulation in high concentrations during estivation helps to minimize the increase in cellular ionic strength that would otherwise occur during desiccation and to alleviate the accompanying negative effects of high salt on individual enzyme activities and overall metabolic regulation.Abbreviations PFK
6-phosphofructo-1-kinase
- PK
pyruvate kinase 相似文献
13.
Properties of pyruvate kinase from soybean nodule cytosol 总被引:2,自引:2,他引:0
The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N2 fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K+ and NH4+ concentrations. In the presence of physiological concentrations of K+ and high phosphoenolpyruvate (PEP) concentrations, NH4+ inhibited enzyme activity. Comparisons of kinetic parameters (Vmax and apparent Ka) for NH4+ and K+ with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent Ka (monovalent cation) and Km (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH4+. 相似文献
14.
A complete kinetic analysis of the forward mitochondrial creatine kinase reaction was conducted to define the mechanism for its rate enhancement when coupled to oxidative phosphorylation. Two experimental systems were employed. In the first, ATP was produced by oxidative phosphorylation. In the second, heart mitochondria were pretreated with rotenone and oligomycin, and ATP was regenerated by a phosphoenolpyruvate-pyruvate kinase system. Product inhibition studies showed that oxidative phosphorylation did not effect the binding of creatine phosphate to the enzyme. Creatine phosphate interacted competitively with both ATP and creatine, and the E · MgATP · CrP dead-end complex was not readily detected. In a similar manner, the dissociation constants for creatine were not influenced by the source of ATP: Kib = 29 mm; Kb = 5.3 mM, and the maximum velocity of the reaction was unchanged: V1 = 1 μmol/ min/mg. Slight differences were noted for the dissociation constant (Kia) of MgATP from the binary enzyme complex, E · MgATP. The values were 0.75 and 0.29 mm in the absence and presence of respiration. However, a 10-fold decrease in the steady-state dissociation constant (Ka) of MgATP from the ternary complex, E · MgATP · creatine, was documented: 0.15 mm with exogenous ATP and 0.014 mm with oxidative phosphorylation. Since Kia × Kb does not equal Ka × Kib under respiring conditions, the enzyme appears to be altered from its normal rapid-equilibrium random binding kinetics to some other mechanism by its coupling to oxidative phosphorylation. 相似文献
15.
Pyruvate kinase (ATP: pyruvate phosphotransferase (EC 2.7.1.40) was partially purified from both autotrophically and heterotrophycally grown Paracoccus denitrificans. The organism grown under heterotrophic conditions contains four times more pyruvate kinase than under autotrophic conditions. The enzyme isolated from both sources exhibited sigmoidal kinetics for both phosphoenolpyruvate (PEP) and ADP. The apparent M
m for ADP and PEP in the autotrophic enzyme were 0.63 mM ADP and 0.25 mM PEP. The effect of several low molecular weight metabolites on the pyruvate kinase activity was investigated. Ribose-5-phosphate, glucose-6-phosphate and AMP stimulated the reaction at low ADP levels; this stimulation was brought about by an alteration in the apparent K
m for ADP. The pyruvate kinases differ in their response to adenine nucleotides, but both preparations seem to be under adenylate control. The results are discussed in relation to the role of pyruvate kinase as a regulatory enzyme in P. denitrificans grown under both autotrophic and heterotrophic conditions.Non-Common Abbreviations PEP
phosphoenolpyruvate
- R-5-P
ribose-5-phosphate
- G-6-P
glucose-6-phosphate
- F-6-P
fructose-6-phosphate
- 3-PGA
3-phosphoglycerate 相似文献
16.
We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na+, K+-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na+, K+-ATPase with II-A and found that inhibitory activity was independent of ATP (2–8 mM), Na+ (3.1–100 mM), or K+ (2.5–40 mM) concentration. Hanes-Woolf plots showed that II-A decreases Vmax in all cases; KM value decreased for ATP but remained unaltered for Na+ and K+, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K+ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K+ concentration. 相似文献
17.
Paramecium tetraurelia expresses four types of arginine kinase (AK1–AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis–Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3–15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R2, AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, VmaxSES?=?30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, VmaxES?=?241.7. The dissociation constant for the SES complex (KiSES: 0.34 mM) was two times smaller than that of the ES complex (KsES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower VmaxSES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the KiSES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, VmaxES and VmaxSES values for the mutants decreased by one-third, except for the VmaxSES of the S79A mutant, which had a value that was comparable with the value for the wild-type. 相似文献
18.
Isolation and Characterization of Phosphoenolpyruvate Phosphatase from Germinating Mung Beans (Vigna radiata) 总被引:15,自引:7,他引:8
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A phosphoenolpyruvate (PEP) phosphatase was purified to homogeneity from germinating mung beans (Vigna radiata). It was found to be a tetrameric protein (molecular mass 240,000 daltons) made up of apparently identical subunits (subunit molecular mass 60,000 daltons). It was free from bound nucleotides. It did not show pyruvate kinase activity. The enzyme showed high specificity for PEP. Pyrophosphate and some esters (nucleoside di- and triphosphates) were hydrolyzed slowly and phosphoric acid monoesters were not hydrolyzed. The enzyme showed maximum activity at pH 8.5. At this pH, the Km of PEP was 0.14 millimolar and the Vmax was equal to 1.05 micromoles pyruvate formed per minute per milligram enzyme protein. Dialysis of the enzyme against 10 millimolar triethanolamine buffer (pH 6.5), led to loss of the catalytic activity, which was restored on addition of Mg2+ ions (Km = 0.12 millimolar). Other divalent metal ions inhibited the Mg2+ -activated enzyme. PEP-phosphatase was inhibited by ATP and several other metabolites. 相似文献
19.
The kinetics of NADP-GPD from spinach chloroplasts are biphasic vs NADPH and PGA. Thus, two maximum velocities exist with an intermediary plateau and two Km values. Activation by NADPH + DTT increases Vmax of both sections, but does not change the substrate affinities. Sulphite reduces the maximum activities of both sections vs NADPH, however, it causes normal substrate kinetics vs PGA; even Vmax is reduced. Sulphite, present only during the activation process, suppresses the enzyme form with the higher Vmax. The kinetics vs NADH are also biphasic; the activity is strongly reduced by preincubation of the chloroplasts with NADH + DTT or at NADH concentrations > 0.4mM. Using NADH as cofactor, inverted peaks in the kinetics vs PGA occur; sulphite is active in a similar way as when NADPH is used as cofactor. The biphasic kinetics are discussed with respect to additional potential for regulation of enzyme activity according to illumination and NADPH concentrations respectively. 相似文献
20.
G E Willick K Oikawa W D McCubbin C M Kay 《Biochemical and biophysical research communications》1973,53(3):923-928
The binding of the fluorescent analog of adenosine diphosphate (ADP)1, 1,N6-ethenoadenosine diphosphate (εADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for εADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 (± .5) × 10?5 M?1. This is identical to the values found by Young (J. Biol. Chem., , 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of εATP versus ATP by S1 were studied. Values of Vmax and Km were 25 μM phosphate sec?1 (gm protein)?1 and 5 × 10?5 M?1 for ATP, and 80 μN phosphate sec?1 (gm protein)?1 and 45 × 10?5 M?1 for εATP. The results indicate that the increased Vmax that occurs when εATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of εATP versus ADP. This in turn suggests that the increase in Vmax may be due to an increased hydrolytic rate of εATP vs ATP in the enzyme substrate complex. 相似文献