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PHOSPHOROLYSIS OF a (1-4)LINKAGES UP TO 4 GLUCOSE RESIDUES FROM AN a (1-6)BRANCH POINT

GLYCOGEN BREAKDOWN. PHOSPHORYLASE. (GLUCOSE)n + Pi (GLUCOSE)n-1 + G-1-P. PHOSPHOROLYSIS OF a (1-4)LINKAGES UP TO 4 GLUCOSE RESIDUES FROM AN a (1-6)BRANCH POINT. GLYCOGEN SYNTHESIS. HEXOKINASE. GLUCOSE + ATP GLUCOSE-6-PHOSPHATE + ADP. PHOSPHOGLUCOMUTASE.

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PHOSPHOROLYSIS OF a (1-4)LINKAGES UP TO 4 GLUCOSE RESIDUES FROM AN a (1-6)BRANCH POINT

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  1. GLYCOGEN BREAKDOWN PHOSPHORYLASE (GLUCOSE)n + Pi (GLUCOSE)n-1 + G-1-P PHOSPHOROLYSIS OF a(1-4)LINKAGES UP TO 4 GLUCOSE RESIDUES FROM AN a(1-6)BRANCH POINT

  2. GLYCOGEN SYNTHESIS HEXOKINASE GLUCOSE + ATP GLUCOSE-6-PHOSPHATE + ADP PHOSPHOGLUCOMUTASE GLUCOSE-6-PHOSPHATE GLUCOSE-1-PHOSPHATE GLUCOSE-1-PHOSPHATE URIDYLTRANSFERASE GLUCOSE-1-PHOSPHATE + UTP UDP-GLUCOSE + PPi GLYCOGEN SYNTHASE UDP-GLUCOSE + GLYCOGEN (n) GLYCOGEN(n+1) + UDP

  3. PHOSPHORYLASE Tower Allosteric effector site N-terminal domain (Glycogen-binding subdomain) N-terminal domain (Interface subdomain) Glycogen Storage site Catalytic site C-terminal domain Pyridoxal phosphate site T conformation (Inactive- Buried active site) R conformation (Active – Accessible Active Site) Stabilized by:AMP Pi Phosphorylation Stabilized by:ATP G-6-P Glucose (Liver)

  4. Pi Pi Pi Pi PHOSPHORYLASE REGULATION PHOSPHORYLASE b KINASE 2ADP 2ATP T FORM T FORM (INACTIVE) (INACTIVE) 2Pi 2H20 SERINE 14 N-TERMINUS (AMP-INDEPENDENT) PROTEIN PHOSPHATASE ATP and/or G-6-P Glucose (Liver) AMP R FORM R FORM (ACTIVE) (ACTIVE) PHOSPHORYLASE a PHOSPHORYLASE b

  5. PHOSPHORYLASE REGULATION BY PHOSPHORYLATION 4ADP 4ATP Ca2+ PHOS a –TETRAMER PHOSPHORYLASE b KINASE 2(PHOS b) DIMER PROTEIN PHOSPHATASE-1 AMP INDEPENDENT P ACTIVATORS:AMP, Pi INHIBITORS:ATP, G-6-P AMP INHIBITS 4Pi GLYCOGEN(n) GLYCOGEN(n-1) + G-1-P

  6. STRUCTURE OF PHOSPHORYLASE KINASE REGULATORY SUBUNITS b b a a ACTIVE SITE d d g g g g CALCIUM-BINDING PROTEIN (CALMODULIN) d d a a b b • ENTIRELY IN b FORM IN RESTING • MUSCLE • ACTIVATED BY: • (i) INCREASED Ca 2+ • - CONTRACTION • - a1-ADRENERGIC AGENTS • - ANGIOTENSIN • - VASOPRESSIN • PHOSPHORYLATION • - cAMP-DEPENDENT PROTEIN • KINASE

  7. PHOSPHORYLASE KINASE REGULATION Ca2+ PHOSPHORYLATED KINASE Ca2+-FREE KINASE Ca2+-KINASE PARTIALLY ACTIVE FULLY ACTIVE Reduced Ca2+ Requirement (a form) 15-20-fold  activity Ca 2+ sensitivity 20mM to 1mM INACTIVE Requires Ca2+ bound to d subunit (b form) cAMP-DEPENDENT PROTEIN KINASE ATP ADP PHOSPHORYLASE b KINASE a (FULLY ACTIVE) PHOSPHORYLASE b KINASE b (LESS ACTIVE) Pi PROTEIN PHOSPHATASE -1 cAMPPK

  8. PI-1 PI-1 PI-1 GBS GBS P P P P PP1 PP1 PP1 DEPHOSPHORYLATION OF PHOSPHORYLASE AND PHOSPHORYLASE KINASE PROTEIN PHOSPHATASE 1 95% ACTIVITY TOWARDS PHOSPHORYLASE 95% ACTIVITY TOWARDS b-SUBUNIT OF PHOSPHORYLASE KINASE 85% ACTIVITY TOWARDS GLYCOGEN SYNTHASE (Sites 1a, 2, 3(a,b,c)) TIGHT COMBINATION WITH GLYCOGEN VIA GLYCOGEN BINDING SUBUNIT. RELEASED ON PHOSPHORYLATION BY cAMPPK (SITES 1 & 2) INHIBITED BY PROTEIN PHOSPHATASE 1 INHIHIBITOR (PI-1) WHEN PI-1 ACTIVATED BY PHOSPHORYLATION BY cAMPPK. GLYCOGEN GLYCOGEN INACTIVE + ACTIVE ACTIVE INACTIVE

  9. PROTEIN PHOSPHATASES TYPE 2 INSENSITIVE TO INHIBITOR PROTEINS. PREFERENTIALLY DEPHOSPHORYLATES a-SUBUNIT OF PHOSPHORYLASE KINASE. TYPE 2A (PP-2A) 36kDa C SUBUNIT, 60kDa A SUBUNIT, 54kDa B SUBUNIT. ACCOUNTS FOR AN APPRECIABLE AMOUNT OF GLYCOGEN SYNTHASE ACTIVITY AND A SMALL AMOUNT OF PHOSPHORYLASE AND PHOSPHORYLASE KINASE ACTIVITY. MAJOR PHOSPHORYLASE KINASE a-SUBUNIT AND PI-1 PHOSPHATASE IN ABSENCE Ca2+. TYPE 2C (PP-2C) DIMER OF 44kDa SUBUNIT. NO ACTIVITY TOWARDS ENZYMES OF GLYCOGEN METABOLISM. TYPE 2B (PP-2B) CONSISTS OF A SUBUNIT (CATALYTIC) AND B SUBUNIT (CALCIUM BINDING). DEPHOSPHORYLATES INHIBITOR-1 AND REGULATORY SUBUNIT OF PKA, a-SUBUNIT OF PHOSPHORYLASE KINASE AND MYOSIN LIGHT CHAIN KINASE. BINDS TO CALMODULIN TO INCREASE ACTIVITY 10-FOLD. INCREASES Vmax. LITTLE ACTIVITY TOWARDS METABOLIC ENZYMES. MAY BE MAJOR PHOSPHATASE DURING CONTRACTION

  10. +G-6-P Glucose Pi PHOSPHATASE GLYCOGEN SYNTHASE D GLYCOGEN SYNTHASE I Less Active Active KINASE ATP ADP TETRAMER Activated by G-6-P Inhibited by ATP, Pi, UDP CONTROL OF GLYCOGEN SYNTHASE NINE PHOSPHORYLATION SITES (SERINE RESIDUES) - SITES 2 AT THE N TERMINUS, REMAINDER AT C TERMINUS. REQUIRE PHOSPHORYLATION AT BOTH N AND C TERMINUS FOR INACTIVATION.

  11. GLYCOGEN SYNTHASE PHOSPHORYLATION CAMPPK 1A > 2 > 1B PHOSPHORYLASE KINASE (Ca2+) 2 GSK 3 3A, 3B, 3C GSK4 2 GSK5 5 CAM DEPENDENT PK (Ca 2+) 2 PHOSPHORYLATION INCREASES THE KM FOR UDP-GLUCOSE. INCREASES KA FOR G-6-P AND DECREASES KI FOR PI. MOST STRONGLY INACTIVATING SITES - 3A,B,C > 2 > 1 EFFECTS ARE ADDITIVE ACTIVITY  IN RESPONSE TO (I) ADRENALINE (II) GLYCOGEN CONTENT OF MUSCLE ACTIVITY  IN RESPONSE TO (I) INSULIN (II) GLYCOGEN CONTENT OF MUSCLE IN FED ANIMAL ACTIVITY LOW AND ENZYME PHOSPHORYLATED CF. PHOSPHORYLASE KINASE AND PHOSPHORYLASE, ACTIVITY LOW AND DEPHOSPHORYLATED - cAMP LOW AND THEREFORE NOT cAMPPK. GSK-3 THE MAJOR KINASE

  12. CONTROL OF GLYCOGEN SYNTHASE BY ADRENALINE AND INSULIN ADRENALINE INHIBITS GLYCOGEN SYNTHASE BY cAMP DEPENDENT MECHANISM ADRENALINE PROPRANOLOL ACTIVITY 20% 100% PHOSPHATE 5MOLES/SUBUNIT 3MOLES/SUBUNIT 50% OF Pi IN SITES 3a,b,c. 50% IN SITES 1a, 1b, AND 2. 75% DECREASE IN ACTIVITY RESULTS FROM SITES 3a,b,c. CATALYSED BY GSK-3 WHICH IS cAMP-INDEPENDENT, THEREFORE MAJOR INACTIVATING EFFECT IS DUE TO INHIBITION OF PHOSPHATASE RATHER THAN ACTIVATION OF KINASE INSULIN TREATMENT REDUCES Pi BY 0.5 MOLES Pi/SUBUNIT FROM SITES 3a,b,c (NOT CAMP MEDIATED) THEREFORE EITHER IT INHIBITS GSK3 OR ACTIVATES PHOSPHATASE ALSO  GLUCOSE TRANSPORT (GLUT4) AND  G-6-P

  13. R2 C2 (Inactive) PP1G P P P P P P (Active) PhK (Less Active) GS (Active) CONTROL OF GLYCOGEN METABOLISM BY cAMP ADRENALINE GLUCAGON (Muscle) (Liver) b-RECEPTOR RECEPTOR ATP cAMP 2C + R2 (cAMP)4 PI-1 (Active) PP2A PI-1 PPI- PI-1 PP1 + G PhK PP2A Sites 1 & 2 (More Active) Sites 1a, 2,1b (Inactive) Site 2 GS (Less Active) Phosa Phos b X (Active) (Inactive)

  14. CONTROL OF GLYCOGEN SYNTHASE BY INSULIN GLUCOSE INSULIN GLUT4 RECEPTOR Glucose IRS-1, SHC Grb2/mSOS RAS PI-3K G-6-P RAF PKB MEK GSK3 MAP KINASE (Inactive) C p90rsk PP1G Site 1 + (More active) Other pathways ? N GLYCOGEN SYNTHASE = (ISPK, MAPKAK-1) p90rsk The relative importance of these pathways may vary from tissue to tissue

  15. PP1G P P P P P P (Active) CONTROL OF GLYCOGEN METABOLISM BY INSULIN GLUCOSE INSULIN Receptor Glut4 TYROSINE KINASE GSK-3 GLUCOSE PKB MAPK (Active) (Active) G-6-P ISPK (p90rsk) ISPK GSK-3 (Active) (Inactive)  PHOSPHORYLASE  GLYCOGEN SYNTHASE PP1G (More Active) GS Site1 (Less Active) X PHOSKa (More Active) GS (More Active) PHOSKb ADRENALINE PPIG –SITES 1 & 2 PHOSa PHOSb (Less Active) (Active) (Inactive)

  16. GLYCOGEN METABOLISM IN LIVER MANY EFFECTS THROUGH Ca2+ RATHER THAN cAMP VIA ADRENALINE (1), ANGIOTENSIN II AND VASOPRESSIN. PHOSPHORYLASE – DIFFERENT ISOENZYME FROM MUSCLE. NOT CONTROLLED BY AMP OR G-6-P. GLUCOSE BINDS TO ACTIVE SITE AND COMPETITIVELY INHIBITS THE ENZYME AND MAKES MORE SUSCEPTIBLE TO DEPHOSPHORYLATION GLYCOGEN SYNTHASE - PHOSPHORYLATION DECREASES Vmax RATHER THAN INCREASING Km FOR UDPG. NO SITES 1a AND 1b. GLUCAGON AND GLUCOSE MAIN EFFECTORS. G-6-P PROMOTES DEPHOSPHORYLATION BY PP1 AS WELL AS ALLOSTERICALLY ACTIVATING. PP1-G – GLYCOGEN BINDING SUBUNIT SMALLER AND NO EVIDENCE FOR CONTROL BY PHOSPHORYLATION. G SUBUNITS ANCHOR PHOSPHATASE TO GLYCOGEN PARTICLES AND DECREASES SENSITIVITY TO PI-1. BINDS STRONGLY TO PHOSa BUT NOT PHOSb

  17. GLYCOGEN METABOLISM IN LIVER 11 GLUCAGON – INCREASES cAMP LEADING TO INCREASED PHOS KINASE AND PHOSa ACTIVITY. PHOSa ACTS AS ALLOSTERIC INHIBITOR TO INHIBIT PPI-G AND PREVENT DEPHOSPHORYLATION. SIMILAR EFFECTS OCCUR WITH ADRENALINE VIA INCREASED Ca2+ AND PHOS KINASE. BOTH PHOSPHORYLATE GLYCOGEN SYNTHASE VIA cAMPPK AND Ca2+/CALMODULIN DEPENDENT PROTEIN KINASES. INSULIN – ACTIVATES cAMP-PDE VIA PI-3-KINASE PATHWAY LEADING TO DECREASE IN cAMP AND REDUCTION IN PHOS KINASE AND PHOSa AND DECREASE IN GLYCOGENOLYSIS. ALSO ATTENUATES THE ALLOSTERIC INHIBITION OF PPI-G CAUSING RELEASE OF PP1-G AND DEPHOSPHORYLATION OF GLYCOGEN SYNTHASE AND PHOSPHORYLASE KINASE. EFFECT OF GLUCOSE – ENHANCES GLYCOGEN DEPOSITION. PHOSa FUNCTIONS AS GLUCOSE RECEPTOR. BINDS TO ACTIVE SITE AND COMPETITIVELY INHIBITS THE ENZYME. ALSO INDUCES CONFORMATIONAL CHANGE (STABILISES THE T STATE) CAUSING SER 14 TO BECOME ACCESSIBLE TO PP1-G LEADING TO DEPHOSPHORYLATION. CAUSES RELEASE OF PP1-G ALLOWING IT TO INTERACT WITH GLYCOGEN SYNTHASE SWITCHING IT ON.

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