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1.
The kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of concentrations of AMP under the saturation of the enzyme by glycogen. The applicability of 23 different variants of the kinetic model involving the interaction of AMP and glucose 1-phosphate binding sites in the dimeric enzyme molecule is considered. A kinetic model has been proposed which assumes: (i) the independent binding of one molecule of glucose 1-phosphate in the catalytic site on the one hand, and AMP in both allosteric effector sites and both nucleoside inhibitor sites of the dimeric enzyme molecule bound by glycogen on the other hand; (ii) the binding of AMP in one of the allosteric effector sites results in an increase in the affinity of other allosteric effector site to AMP; (iii) the independent binding of AMP to the nucleoside inhibitor sites of the dimeric enzyme molecule; (iv) the exclusive binding of the second molecule of glucose 1-phosphate in the catalytic site of glycogen phosphorylase b containing two molecules of AMP occupying both allosteric effector sites; and (v) the catalytic act occurs exclusively in the complex of the enzyme with glycogen, two molecules of AMP occupying both allosteric effector sites, and two molecules of glucose 1-phosphate occupying both catalytic sites. 相似文献
2.
An allosteric model of phosphorylase B is proposed based on the following assumptions. The enzyme consists of two sub-units and undergoes a concerted transition from the inactive T to the active R state. The binding of substrates, phosphate, and glycogen is regarded as exclusive, but the binding of the activator AMP is nonexclusive. The enzyme model is of the K, V type, i. e., the activator AMP is important, not only for the T-R transition and the substrates binding, but also for the formation of the active site. Therefore, it displays a big influence on the maximal reaction rate. Calculations based on this model lead to an equation containing 5 constants, which can be easily computed from kinetic data. All kinetic measurements fit the expressions derived from the model. Independent methods for the measurement of all the constants involved were developed. They are based on the study of binding of phosphorylase with the substrates and the activator. These measurements are in satisfactory agreement with the data obtained from enzyme kinetics. 相似文献
3.
YOSHINORI MUTO SHUZO KUDO YOSHINORI NOZAWA 《The Journal of eukaryotic microbiology》1984,31(1):164-166
Glycogen phosphorylase in Tetrahymena pyriformis was activated by a Mg2+ ATP-dependent process and this activation was further increased by the addition of cyclic AMP. When the enzyme activity in subcellular fractions was measured, it was largely associated with the glycogen fraction but was no longer activated by ATP and cyclic AMP. Mixing the glycogen fraction and cytosol fraction together restored the effects of ATP and cyclic AMP on phosphorylase activity. These findings suggest that glycogen phosphorylase associated with Tetrahymena glycogen granules may be regulated by cytosolic factor(s) with cyclic AMP. 相似文献
4.
S V Klinov N A Chebotareva B M She?man E M Birinberg B I Kurganov 《Bioorganicheskaia khimiia》1987,13(7):908-914
The interaction of rabbit skeletal muscle glycogen phosphorylase b with methotrexate, folic and folinic acids has been studied. Microscopic dissociation constant for the glycogen phosphorylase b--methotrexate complex determined by analytical ultracentrifugation is 0.43 mM. A subunit of glycogen phosphorylase b is shown to have two sites for methotrexate binding. AMP and FMN diminish the affinity of glycogen phosphorylase b to methotrexate, whereas glycogen does not influence the methotrexate binding to the enzyme. Methotrexate, folic and folinic acids are found to be inhibitors of the muscle glycogen phosphorylase b. The inhibition is reversible and characterized by positive kinetic cooperativity (the Hill coefficient exceeds one unity). The value of the pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: folic acid (0.65 mM), methotrexate (1.01 mM), folinic acid (3.7 mM). The antagonism between methotrexate, folic and folinic acids, on the one hand, and AMP and FMN, on the other, is revealed for their combined action. 相似文献
5.
P A Santiago D Santiago A M Block F Sagardía 《Archives of biochemistry and biophysics》1974,163(2):688-698
The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with Pi or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to Pi changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to Pi. Arsenate is a competitive inhibitor with respect to Pi. The Km for AMP increases in the presence of UDPG, and decreases with increasing concentrations of Pi or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for Pi, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors. 相似文献
6.
JosM. Bautista Amando Garrido-Pertierra Germn Soler 《Biochimica et Biophysica Acta (BBA)/General Subjects》1988,967(3)
The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies,NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP+ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903–904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP+] ratio. 相似文献
7.
S. Kawasaki Watanabe G. Hernandez-Velazco F. Iturbe-Chiñas A. Lopez-Munguia 《World journal of microbiology & biotechnology》1992,8(4):406-410
Simultaneous depletion of phenylalanine and tyrosine by phenylalanine ammonia lyase is described in a mutual competitive inhibition model. The enzymes obtained fromSporidiobolus pararoseus andRhodosporidium toruloides were charaterized in terms of stability, optimal reaction parameters and kinetic behaviour. Both enzymes followed Michaelis-Menten kinetics with respect to the two amino acids. However, the enzyme fromRhodosporidium toruloides was inhibited by high tyrosine concentrations. 相似文献
8.
Cho KM Lim WJ Math RK Asraful Islam SM Hong SJ Kim H Yun HD 《Journal of molecular evolution》2008,67(1):1-12
A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Omega mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. gamma-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed. 相似文献
9.
Natalija P. Sugrobova 《Journal of biochemical and biophysical methods》1983,8(4):299-306
A turbidimetric method has been developed for the continous monitoring of the enzyme reaction catalyzed by glycogen phosphorylase. This method is based on the registration of the turbidity of glycogen solution at wavelengths above 300 nm. It has been shown that increase in the turbidity is strictly proportional to the quantity of glucose 1-phosphate formed during the enzyme reaction. The method has the advantage of continuity, and it is suitable for determining the initial rate of catalytic synthesis or degradation of glycogen in a relatively simple and fast way. The kinetic experiments may be carried out under various conditions. The method of calculation of the overall equilibrium constant of the enzyme reaction catalyzed by glycogen phosphorylase has been elaborated. This method is based on the analysis of the dependence of the initial rate of the enzyme reaction on the proportiona of the substrate of the forward reaction: [Pi]/([Pi]+[G-1-P]). 相似文献
10.
Interest in the kinetics of glycogen phosphorylase has recently been renewed by the hypothesis of a glycogen shunt and by the potential of altering phosphorylase to treat type II diabetes. The wealth of data from studies of this enzyme in vitro and the need for a mathematical representation for use in the study of metabolic control systems make this enzyme an ideal subject for a mathematical model. We applied a two-part approach to the analysis of the kinetics of glycogen phosphorylase b (GPb). First, a continuous state model of enzyme-ligand interactions supported the view that two phosphates and four ATP or AMP molecules can bind to the enzyme, a result that agrees with spectroscopic and crystallographic studies. Second, using minimum error estimates from continuous state model fits to published data (that agreed well with reported error), we used a discrete state model of internal molecular events to show that GPb exists in three discrete states (two of which are inactive) and that state transitions are concerted. The results also show that under certain concentrations of substrate and effector, ATP can activate the enzyme, while under other conditions, it can competetively inhibit or noncompetitively inhibit the enzyme. This result is unexpected but is consistent with spectroscopic, crystallographic, and kinetic experiments and can explain several previously unexplained phenomena regarding GPb activity in vivo and in vitro. 相似文献
11.
A. R. Venugopala Reddy C. V. Balakrishnan J. Sobhanaditya S. D. Ravindranath V. S. Ananthanarayanan N. Appaji Rao 《Journal of biosciences》1980,2(3):211-225
A homogenous and crystalline form of nucleotide pyrophosphatase (EC 3.6.1.9) fromPhaseolus aureus (mung bean) seedlings was used for the study of the regulation of enzyme activity by adenine nucleotides. The native dimeric
form of the enzyme had a helical content of about 65% which was reduced to almost zero values by the addition of AMP. In addition
to this change in the helical content, AMP converted the native dimer to a tetramer. Desensitization of AMP regulation, without
an alteration of the molecular weight, was achieved either by reversible denaturation with 6 M urea or by passage through
a column of Blue Sepharose but additionofp-hydroxymercuribenzoate desensitized the enzyme by dissociating the native dimer to a monomer. The changes in the quaternary
structure and conformation of the enzyme consequent to AMP interaction or desensitization were monitored by measuring the
helical content, EDTA inactivation and Zn2+ reactivation, stability towards heat denaturation, profiles of urea denaturation and susceptibility towards proteolytic digestion.
Based on these results and our earlier work on this enzyme, we propose a model for the regulation of the mung bean nucleotide
pyrophosphatase by association-dissociation and conformational changes. The model emphasizes that multiple mechanisms are
operative in the desensitization of regulatory proteins. 相似文献
12.
M P Leckie R H Ng S E Porter D R Compton D N Dietzler 《The Journal of biological chemistry》1983,258(6):3813-3824
In Escherichia coli cya mutants, deficient in adenylate cyclase (EC 4.6.1.1), basal cellular rates of glycogen synthesis were lower and the relative increases produced by exogenous cyclic adenosine 3',5'-monophosphate during growth on glucose were greater than in their respective parent strains. These observations provide strong evidence that endogenous cyclic AMP is one of the key regulators of glycogen synthesis in growing E. coli. In crp mutants, deficient in cyclic AMP receptor protein (CRP), the basal cellular rates of glycogen synthesis were much lower than in their respective parent strains. Stimulation of glycogen synthesis by exogenous cyclic AMP was markedly attenuated in the three crp mutants. Thus, stimulation of glycogen synthesis by either endogenous or exogenous cyclic AMP appears to require CRP. Functional CRP appeared to be required for all three responses observed after cyclic AMP addition: an abrupt step-up in the cellular rate of glycogen synthesis, a continuing exponential increase in rate, and a stimulation of the rate during a subsequent nitrogen starvation. To account for these responses, we derived a mathematical model in which the cyclic AMP-CRP complex regulates the differential rate of synthesis of an enzyme metabolizing an effector of the rate-limiting enzyme of glycogen synthesis. 相似文献
13.
Enzymatic depolymerization of polysaccharides with alpha-amylase has been studied in mixed aqueous dimethylsulfoxide (DMSO)/water solvents. Polysaccharide substrate chemical compositions, configurational structures, and bonding pattersn are known to affect observed enzymatic reaction kinetics. The branching structures of polysaccharides and their effects on the kinetic mechanisms of depolymerization reactions via endo-acting hydrolyzing enzyme was studied via size exclusion chromatography coupled to low angle laser light scattering (SEC/LALLS). The glycogen branching structure is a heterogeneously distributed "cluster" structure rather than a homogeneously distributed "treelike" structure. The action pattern of alpha-amylase on glycogen, which is composed of highly branched clusters, as end-products, has a "pseudo-exo-attack" in contrast to an expected "endoattack" as seen in the hydrolysis of amylose or amylopectin substrates. These effects of branched substrates for mixed amylose/glycogen alpha-amylolysis have been predicted and demonstrated by both experimental and theoretical analysis using the kinetic model presented in this report. The "lumped" kinetic model employed, assumes that the enzyme simultaneously attacks both linear and branched substrates. In general, excellent agreement between the model predictions and the experimental observations, both qualitatively and quantitatively, was obtained. (c) 1995 John Wiley & Sons, Inc. 相似文献
14.
Summary The kinetic constants for the series of glucosyl acceptors for homogeneous rabbit muscle glycogen synthaseI form free of glycogen were examined. The acceptors included glucose, maltose, G3, G4, G6, two hydrolyzed amyloses, amylodextrin and seven polysaccharides including amylopectin and glycogen. S0.5 and relative Vmax were estimated in each case. From these data a two site model of the enzyme is proposed, composed of a polysaccharide binding site and a separate catalytic site, the latter composed of several subsites. 相似文献
15.
The regulatory properties of NADH-dependent nitrate reductase (NR) in desalted root extracts from hydroponically grown cucumber
(Cucumis sativus L.) seedlings were examined. The lowest activity of NR was detected in extracts incubated with Mg2+ and ATP. An inhibitory effect of Mg-ATP was cancelled in the presence of staurosporine (the protein kinase inhibitor) and
completely reversed after addition of ethylenediaminetetraacetate (EDTA) as well as AMP into reaction mixture. Reactivation
of enzyme due to AMP presence, contrary to the chelator-dependent NR activation, was sensitive to microcystin LR (the protein
phosphatase inhibitor). Above results indicated that the nitrate reductase in cucumber roots was regulated through reversible
phosphorylation of enzyme protein. A drop in the activity of NR was also observed after incubation of enzyme at low pH. At
low pH, the presence of ATP alone in the incubation medium was sufficient to inactivate NR, indicating that H+ can substitute the Mg2+ in formation of an inactive complex of enzyme. ATP-dependent inactivation of NR at low pH was prevented by staurosporine
and reversed by AMP. However, AMP action was not altered by microcystin LR suggesting that in low pH the nucleotide induced
reactivation of NR is not limited to the protein phosphorylation. 相似文献
16.
Stephen P. J. Brooks Kenneth B. Storey 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(1):23-28
Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P2) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M1-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M2 isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P2 may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP
adenosine diphosphate
- AMP
adenosine monophosphate
- ATP
adenonine triphoshate
- EDTA
ethylenediaminetetra-acetic acid
- EGTA
ethylene glycol tetra-acetic acid
- F-6-P
fructose 6-phosphate
- F-1,6-P2
fructose 1,6-bisphosphate
- F-2,6-P2
fructose-2,6-bisphosphate
-
K
a
activation coefficient
- I50
concentration of inhibitor which reduces control activity by 50%
- PDH
pyruvate dehydrogenase
- PEP
phosphoenolpyruvate
- PFK
6-phosphofructo-1-kinase
- PK
pyruvate kinase 相似文献
17.
Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate + ATP + CoA Acetyl-CoA + AMP + PPi). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41°C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37°C. Using the N-terminal amino acid sequence an open reading frame — coding for a 74 kDa protein — was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS. 相似文献
18.
Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens. 总被引:4,自引:1,他引:3
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1.The two forms of glycogen phosphorylase were purified from human liver, and some kinetic properties were examined in the direction of glycogen synthesis. The b form has a limited catalytic capacity, resembling that of the rabbit liver enzyme. It is characterized by a low affinity for glucose 1-phosphate, which is unaffected by AMP, and a low V, which becomes equal to that of the a form in the presence of the nucleotide. Lyotropic anions stimulate phosphorylase b and inhibit phosphorylase a by modifying the affinity for glucose 1-phosphate. Both enzyme forms are easily saturated with glycogen. 2. These kinetic properties have allowed us to design a simple assay method for total (a + b) phosphorylase in human liver. It requires only 0.5 mg of tissue, and its average efficiency is 90% when the enzyme is predominantly in the b form. 3. The assay of total phosphorylase allows the unequivocal diagnosis of hepatic glycogen-storage disease caused by phosphorylase deficiency. One patient with a complete deficiency is reported. 4. The assay of human liver phosphorylase a is based on the preferential inhibition of the b form by caffeine. The a form displays the same activity when measured by either of the two assays. 相似文献
19.
Interaction of flavin mononucleotide with dimeric and tetrameric forms of muscle phosphorylase beta.
Interaction of flavin mononucleotide (FMN) with dimeric and tetrameric forms of rabbit muscle glycogen phosphorylase beta has been studied under the conditions when allosteric activator binding sites are saturated by AMP (1 mM AMP; pH 6.8; 17 degrees C). Simultaneous use of schlieren optical system and photoelectric scanning absorption optical system of analytical ultracentrifuge Spinco, model E, makes it possible to register the oligomeric state of the enzyme and calculate the degree of saturation of individual oligomeric enzyme forms by FMN. The apparent association constant for the equilibrium dimer in equilibrium with tetramer decreased with increasing FMN concentration. The microscopic dissociation constants for the complexes of dimeric and tetrameric forms of glycogen phosphorylase beta with FMN have been found to be equal to 10 and 79 microM, respectively. 相似文献
20.
A model of carbohydrate metabolism during differentiation in Dictyostelium discoideum has been used to investigate which enzyme kinetic mechanism(s) might be operative for glycogen phosphorylase in vivo. The model, which has been described previously, is capable of simulating experimentally observed changes in metabolite concentrations and fluxes during differentiation under both the standard starvation condition and in the presence of glucose (25 mM). The concentrations of saccharide end products of differentiation under these 2 conditions differ substantially.Glycogen phosphorylase is described in the model by a rapid equilibrium random bi bi mechanism and the effect of substituting 4 other kinetic mechanisms was examined. Each of these mechanisms in the model allows simulations compatible with the saccharide accumulation patterns found during differentiation in the absence of glucose. However, in the presence of glucose, only a reversible mechanism (random or ordered) is compatible with the experimental data. It is concluded that glycogen degradation in vivo is controlled by an enzyme catalyzing a reversible reaction, the rate of which is inversely related to the glucose-1-P concentration. 相似文献