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PubMed1984

Nonactivated phosphorylase kinase is a phosphoprotein: differentiation of two classes of endogenous phosphoserine residues by phosphorus-31 nuclear magnetic resonance spectroscopy and phosphatase sensitivity.

A standard preparation of phosphorylase kinase from rabbit skeletal muscle contains 2 mol of phosphoserine/mol of alpha beta gamma delta. This basal stoichiometry is not influenced by application of propranolol and insulin in vivo; these serine phosphates could not be hydrolyzed by phosphatases of the muscle extract or by alkaline phosphatases. When the enzyme is purified in the presence of the protein phosphatase inhibitor sodium fluoride, it contains either 1 or 3 additional mol of phosphoserine/mol of alpha beta gamma delta, termed phosphatase-sensitive phosphates. Both classes of phosphates yield in formic acid one single 31P NMR signal of a narrow line width (approximately 3 Hz) very similar in chemical shift to free phosphoserine. Phosphoserine is also identified by its chemical shift when dissolved in 8 M guanidinium chloride and by its electrophoretic mobility after acid hydrolysis. By self-phosphorylation of phosphorylase kinase, 14 additional mol of phosphate/mol of alpha beta gamma delta was incorporated, and all were identified as phosphoserine by 31P NMR spectroscopy. In native phosphorylase kinase, the 31P NMR signals of both the basal and the phosphatase-sensitive phosphates are substantially broadened and reduced in intensity, indicating strong interactions of the phosphate groups with the protein. The basal and phosphatase-sensitive phosphates give in 8 M guanidinium chloride a homogeneous NMR signal above pH 6; it splits into a doublet below pH 6 and into a triplet below pH 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1980

Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions.

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.

Animals↗
PubMed1997

Purine nucleoside phosphorylase. 2. Catalytic mechanism.

X-ray crystallography, molecular modeling, and site-directed mutagenesis were used to delineate the catalytic mechanism of purine nucleoside phosphorylase (PNP). PNP catalyzes the reversible phosphorolysis of purine nucleosides to the corresponding purine base and ribose 1-phosphate using a substrate-assisted catalytic mechanism. The proposed transition state (TS) features an oxocarbenium ion that is stabilized by the cosubstrate phosphate dianion which itself functions as part of a catalytic triad (Glu89-His86-PO4=). Participation of phosphate in the TS accounts for the poor hydrolytic activity of PNP and is likely to be the mechanistic feature that differentiates phosphorylases from glycosidases. The proposed PNP TS also entails a hydrogen bond between N7 and a highly conserved Asn. Hydrogen bond donation to N7 in the TS stabilizes the negative charge that accumulates on the purine ring during glycosidic bond cleavage. Kinetic studies using N7-modified analogs provided additional support for the hydrogen bond. Crystallographic studies of 13 human PNP-ligand complexes indicated that PNP uses a ligand-induced conformational change to position Asn243 and other key residues in the active site for catalysis. These studies also indicated that purine nucleosides bind to PNP with a nonstandard glycosidic torsion angle (+anticlinal) and an uncommon sugar pucker (C4'-endo). Single point energy calculations predicted the binding conformation to enhance phosphorolysis through ligand strain. Structural data also suggested that purine binding precedes ribose 1-phosphate binding in the synthetic direction whereas the order of substrate binding was less clear for phosphorolysis. Conservation of the catalytically important residues across nucleoside phosphorylases with specificity for 6-oxopurine nucleosides provided further support for the proposed catalytic mechanism.

Amino Acid Sequence↗
PubMed2004

Kinetic studies of AMP-dependent phosphorolysis of amylopectin catalyzed by phosphorylase b on a 27 MHz quartz-crystal microbalance.

Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.

Adenosine Monophosphate↗
PubMed1987

Inhibition of intestinal pyrimidine nucleoside phosphorylases.

The activity of 5'-deoxy-5-fluorouridine (dFUR) depends on its activation to 5-fluorouracil (FU) by pyrimidine nucleoside phosphorylases. These enzymes are found in tumors and normal tissues, with the highest activity in the small intestines. The present study examined the inhibition of dFUR phosphorolysis in intestinal tissues. dFUR metabolism in intestinal homogenates was inhibited by uracil (U), uridine (UR), and thymidine (TdR), which are the normal substrates for the phosphorylases. Conversely dFUR reduced the metabolism of these inhibitors. A good agreement was found between the observed data and the computer-fitted data using the equations for competitive inhibition between dFUR and the inhibitors. In the absence of inhibitors, the Vmax of dFUR phosphorolysis was 47.1 +/- 4.9 microM/min and the apparent Km was 910 +/- 167 microM. The Vmax was unaltered by the inhibitors, while the Km was increased with increasing inhibitor concentrations. The maximal inhibition of dFUR metabolism by UR and TdR was about 80%. The Ki's were 372 microM for U, 87.2 microM for UR, and 112 microM for TdR and are orders of magnitude higher than their reported endogenous serum concentrations. The rate of dFUR phosphorolysis to FU in the intact intestinal epithelial crypt cells, indicated by the ratio of FU to dFUR in the intracellular fluid, was reduced by UR in a concentration-dependent fashion. These data indicate that the naturally occurring pyrimidines inhibit competitively the dFUR metabolism by the intestinal phosphorylases, that this inhibition occurs at concentrations much higher than the circulating endogenous levels, and that phosphorolysis is the major route of dFUR metabolism.

Animals↗
PubMed2005

FR258900, a novel glycogen phosphorylase inhibitor isolated from Fungus No. 138354. I. Taxonomy, fermentation, isolation and biological activities.

FR258900 is a novel glycogen synthesis activator produced by Fungus No. 138354. This compound was isolated from the culture broth by solvent extraction and reverse-phase column chromatography. FR258900 stimulated glycogen synthesis and glycogen synthase activity in primary rat hepatocytes. FR258900 exhibited a potent inhibitory effect on the activity of liver glycogen phosphorylase, suggesting that this compound may activate hepatic glycogen synthesis via glycogen phosphorylase inhibition. Thus, this glycogen phosphorylase inhibitor may be useful in the treatment of postprandial hyperglycemia in type 2 diabetes.

Chromatography, High Pressure Liquid↗
PubMed2004

Modulation of thymidine phosphorylase by neoadjuvant chemotherapy in primary breast cancer.

The combination effect of docetaxel and capecitabine on tumour response rate and survival was demonstrated recently in metastatic breast cancer patients. This combination was based on an experimental hypothesis that taxane can increase tumour sensitivity to the effect of capecitabine through the upregulation of thymidine phosphorylase (TP), which is responsible for the metabolism of 5-fluorouracil (5-FU) and its derivatives, including capecitabine. To examine the alteration in TP expression before and after neoadjuvant chemotherapy, 92 patients with primary breast cancer (T2-4N0-1M0) were enrolled in this study; 14 were treated with adriamycin and cyclophosphamide (AC) or epirubicin and cyclophosphamide (EC); 58 with 5-FU, adriamycin, and cyclophosphamide (FAC) or 5-FU, epirubicin, and cyclophosphamide (FEC); and 20 with FEC followed by docetaxel/taxotere (TXT-containing regimen). Thymidine phosphorylase upregulation was seen in 54.4% and 32.6% of patients in tumour cells and stromal cells, respectively. Increases in TP expression were found only in the AC/EC and TXT-containing regimen groups. In conclusion, it was strongly suggested that unlike 5-FU-containing regimens, the taxane and AC combination therapies upregulate TP expression in primary breast cancer. Thymidine phosphorylase upregulation by several anticancer drugs implies the importance of individualised strategies for sensitisation of tumour tissues to 5-FU and its derivatives.

Adult↗
PubMed1999

Influence of substrates on in vitro dephosphorylation of glycogen phosphorylase a by protein phosphatase-1.

The kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the dephosphorylation of phosphorylase a by protein phosphatase-1 (ppase-1). On the basis of the kinetic equation of the substrate reaction in the presence of ppase-1, all the inactivation rate constants for the free enzyme and the enzyme-substrate(s) complexes have been determined. Binding of the allosteric substrate, glucose 1-phosphate, to one subunit of phosphorylase a protects completely against ppase-1 action on either the same subunit or the adjacent subunit, whereas binding of the non-allosteric substrate, glycogen, to one subunit protects this subunit partially, but has no effect on the modification on the neighbouring subunit. Analysis of the data suggests that the allosteric behaviour of phosphorylase a can be interpreted in terms of a modified concerted model. The present method also provides a novel approach for studying dephosphorylation reactions. Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified, this new method would be particularly useful when the regulatory mechanism of the reversible phosphorylation reaction toward certain enzymes is being assessed.

Catalysis↗
PubMed2004

Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio.

A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with alpha-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce beta-D-glucosyl-(1-->4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-beta-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k(0)=5.5 s(-1), K(m)=2.0 mM; synthetic reaction, k(0)=10 s(-1), K(m)=14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.

Amino Acid Sequence↗
PubMed1985

Control of glycogen phosphorylase interconversion by phorbol esters, diacylglycerols, Ca2+ and hormones in isolated rat hepatocytes.

In isolated rat hepatocytes: phosphorylase activation by the ionophore A23187 was enhanced in the presence of tumour-promoting phorbol esters and 1,2- (but not 1,3-) diacylglycerols (dioleoyl- and oleoylacetyl-glycerol), with a similar dose-dependency; the activation of phosphorylase by phenylephrine (1 microM) (but not by vasopressin or glucagon) was inhibited both by tumour-promoting phorbol esters and diacylglycerols, but with a different dose-dependency: complete inhibition was achieved with concentrations of phorbol esters two orders of magnitude lower than those of diacylglycerol; binding of the alpha 1-adrenergic antagonist [3H]prazosin and its displacement by unlabelled prazosin was not significantly affected in the presence of the phorbol esters. The possible involvement of protein kinase C in the control of phosphorylase interconversion is discussed.

Animals↗
PubMed1987

High-affinity binding of glycogen-synthase phosphatase to glycogen particles in the liver. Role of glycogen in the inhibition of synthase phosphatase by phosphorylase a.

1. Post-mitochondrial supernatants were prepared from the livers of 24 h-fasted rats. Upon centrifugation at high speed, the major part of the glycogen-synthase phosphatase activity sedimented with the microsomal fraction. However, two approaches showed that the enzyme was associated with residual glycogen rather than with vesicles of the endoplasmic reticulum. Indeed, the activity was entirely solubilized when the remaining glycogen was degraded either by glucagon treatment in vivo or by alpha-amylolysis in vitro. No evidence could be found for an association of glycogen-synthase phosphatase with the smooth endoplasmic reticulum, as isolated with the use of discontinuous sucrose gradients. 2. After solubilization by glucagon treatment in vivo, synthase phosphatase could be transferred to glycogen particles with very high affinity. Half-maximal binding occurred at a glycogen concentration of about 0.25 mg/ml, whereas glycogen synthase and phosphorylase required 1.5-2 mg/ml. 3. In gel-filtered extracts prepared from glycogen-depleted livers, the activation of glycogen synthase was not inhibited at all by phosphorylase alpha. The inhibition was restored when the liver homogenates were prepared in a glycogen-containing buffer. The effect was half-maximal at a glycogen concentration of about 0.25 mg/ml, and virtually complete at 1 mg/ml. These findings explain long-standing observations that in fasted animals the liver contains appreciable amounts of both synthase and phosphorylase in the active form.

Animals↗
PubMed1989

Rapid activation of glycogen phosphorylase by steroid hormones in cultured rat hepatocytes.

Testosterone (40-300 microM), oestradiol (20-500 microM), progesterone (20-500 microM), dexamethasone (10 nM-1 microM) and corticosterone (1-10 microM) activate glycogen phosphorylase rapidly when added directly to hepatocytes. The activation of phosphorylase was concentration-dependent and occurred after 10 min for dexamethasone, 30 min for testosterone and 60 min for oestradiol and progesterone. This rapid effect does not appear to be dependent on a stimulation of protein synthesis, it is independent of an increase in cyclic AMP, and it is not diminished by the presence of ornithine decarboxylase inhibitors. The stimulation of phosphorylase activity is diminished by depleting the incubation medium of Ca2+ in the presence of 0.5 mM-EGTA, and therefore it may involve changes in the distribution of Ca2+ in the hepatocytes. These results may explain some of the pharmacological effects of sex steroids, and also might contribute to the physiological actions of glucocorticoids.

Animals↗
PubMed1990

Cyclic AMP-dependent protein kinase in mammary tissue of the lactating rat. Activity ratio and responsiveness of the target enzymes acetyl-CoA carboxylase and glycogen phosphorylase to beta-adrenergic stimulation.

The role of cyclic AMP in acute regulation of the metabolism of mammary tissue in the lactating rat was examined by measuring the activity ratio of cyclic AMP-dependent protein kinase (A-kinase) and by examining the properties of this enzyme in its two major isoenzymic forms. Isoenzyme II is the major form in soluble extracts of rat mammary tissue. A-kinase activity ratio in such extracts is unaffected by starvation of the lactating rat. Treatment of the intact rat with isoprenaline, or addition of isoprenaline to incubations in vitro of mammary acini, resulted in a major increase in the activity ratio of A-kinase. These treatments equally affected isoenzymes I and II. The treatment in vitro lead to a rapid depletion of A-kinase as subsequently measured in extracts of acini. The degree of activation of the enzymes acetyl-CoA carboxylase and glycogen phosphorylase in extracts of mammary tissue and of acini was assessed as a function of these treatments. The increased activation of A-kinase induced by isoprenaline was unaccompanied by significant changes in the activity of acetyl-CoA carboxylase in acini, although we previously showed that this agent activates acetyl-CoA carboxylase in intact mammary tissue. Contrastingly, isoprenaline-induced enhancement of A-kinase activity was accompanied by an increase in the activity ratio of phosphorylase in acini. These results indicate that: (a) a normal response of expressed A-kinase activity to cyclic AMP operates in mammary acini and mammary tissue from lactating rats; (b) rapid modulation of the total amount of soluble A-kinase is mediated in mammary epithelial cells by cyclic AMP; (c) phosphorylase, an ultimate target of the protein phosphorylation cascade initiated by A-kinase, is activated in acini under conditions where A-kinase activity is enhanced; and (d) mechanisms other than that of the A-kinase phosphorylation/inhibition model for acetyl-CoA carboxylase regulation must operate in mammary tissue preparations and in vivo to account for the response of this enzyme to enhanced A-kinase activity.

Acetyl-CoA Carboxylase↗
PubMed1990

The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin.

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, beta- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, beta- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only beta-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.

Animals↗
PubMed1991

The binding of D-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies.

Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring.

Amino Acid Sequence↗
PubMed1994

Baculovirus-directed expression of the gamma-subunit of phosphorylase kinase: purification and calmodulin dependence.

A recombinant baculovirus containing a cDNA encoding the gamma-subunit of phosphorylase kinase from mouse skeletal muscle was constructed. Cultures of Sf-9 insect cells infected with the gamma-baculovirus produce an intact and soluble gamma-protein. A purification procedure is presented that yields a sample of gamma-protein which is devoid of interfering enzyme activity and which is not associated with calmodulin from the insect cells. The isolated gamma sample has a Km for phosphorylase b of 36 (+/- 6, S.E.M) microM at pH 8.2 and 140 (+/- 25) microM at pH 6.8. These values are similar to those reported for the activated phosphorylase kinase holoenzyme isolated from skeletal muscle tissue. However, the Vmax. of the baculovirus-expressed gamma is 65 and 80% of that of the activated holoenzyme at pH 6.8 and 8.2 respectively. These results indicate that one or more of the regulatory subunits alpha, beta, or calmodulin stimulate the activity of the catalytic subunit gamma in the activated holoenzyme. Addition of calmodulin to the baculovirus-expressed gamma stimulates its activity 1.5-2.0 fold at pH 6.8 in both the presence and absence of calcium. At pH 8.2, calmodulin has only minor stimulatory affects. The stimulation by calmodulin at pH 6.8 results from an increase in the Vmax of gamma with little effect on its Km. This result is unlike that for most calmodulin-stimulated kinases which bind calmodulin only in the presence of calcium and exhibit a decrease in their Km upon binding calmodulin. The change in Vmax. of gamma in the presence of calmodulin and in the absence of calcium presents a novel mechanism for the regulation of a calmodulin-stimulated kinase.

Animals↗
PubMed1996

Measurement of the turnover of glycogen phosphorylase by GC/MS using stable isotope derivatives of pyridoxine (vitamin B6).

The majority of vitamin B6 in the body is in skeletal muscle, bound as the cofactor pyridoxal 5'-phosphate to one abundant protein, glycogen phosphorylase. Previous work has established that radiolabelled vitamin B6 can be used as a turnover label for glycogen phosphorylase. In this study, a stable isotope derivative of pyridoxine {dideuterated pyridoxine; 3-hydroxy-4-(hydroxymethyl) -5-[hydroxymethyl-2H2]-2-methylpyridine} ([2H2]PN) has been used as a metabolic tracer to study the kinetics of labelling of the body pools of vitamin B6 in mice. A non-invasive method was developed in which the isotope abundance of the urinary excretory product of vitamin B6 metabolism, 4-pyridoxic acid, was analysed by GC/MS. The change in isotope abundance of urinary 4-pyridoxic acid following administration of [2H2]PN reflects the kinetics of labelling of the body pools of vitamin B6, and yields, non-invasively, the rate of degradation of glycogen phosphorylase.

Animals↗
PubMed1997

Kinetic mechanism of the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis: co-operative interactions of AMP and glucose 1-phosphate during catalysis.

We employed our newly developed, continuous, spectrophotometric method [Sergienko and Srivastava (1994) Anal. Biochem. 221, 348-355] for measuring the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis, utilizing varied concentrations of AMP (2-400 microM) and glucose 1-phosphate (G1P; 4 microM to 41 mM). The experimental data revealed that the enzyme catalysis exhibits sigmoidal dependence on both AMP and G1P concentrations, with Hill coefficient and EC50 values (mutually) affected by the concentrations of the above substrates. A detailed kinetic analysis of the substrate-dependent activation, as well as glucose-inhibition data, lead us to propose the following mechanistic features of the glycogen-phosphorylase-catalysed reaction. (1) The enzyme exhibits catalytic activity when two molecules of AMP and two molecules of G1P are bound to the dimeric unit. (2) The binding of one molecule of glucose (the competitive inhibitor of G1P) per dimeric unit results into a complete loss of the enzyme activity. (3) There is no restriction of binding of AMP or G1P when one of the dimeric subunits is already bound with the other ligand. For example, one or two G1P molecules can bind to the enzyme dimer when zero, one or two molecules of AMP are already bound. The magnitudes of rate and equilibrium constants for the glycogen-phosphorylase-catalysed reaction, derived from analyses of the experimental data in the light of a few selected minimal models, are presented.

Adenosine Monophosphate↗