Biochem Exam III Flashcards

Question

How is ATP produced from ATP synthase? What is the overall reaction and the steps?

Answer 1

Overall reaction: ADP(3-) + HPO4(2-) ---> ATP(4-) + H2O Steps 1) A single proton from the inner mitochondrial membrane space (cytosol) enters the ATP synthase complex via an F0 a-subunit, hydrophilic, cytoplasmic half-channel 2) The proton in the F0 a-subunit half channel protonates Asp 61 on one of the c-subunits on the c-subunit ring and neutralizes the negative charge 3) When the Asp 61 charge is neutralized, the uncharged Asp 61 can be exposed to a hydrophobic interior of the inner mitochondrial membrane meaning the c ring can rotate clockwise. This then exposes the next charged Asp 61 to a proton on the a-subunit from the cytosol 4) After all of the c subunits become protonated, when one c subunit rotates back to the F0 subunit, the Asp6 becomes deprotonated and a proton is released into the mitochondrial matrix via the other F0 a-subunit half channel. It is then protonated via the a-subunit channel containing a proton from the cytosol and the C ring can spin 5) The clockwise rotation of the c-subunit causes the y-subunit core to rotate within the a3B3 hexamer 6) As the y-subunit rotates the three B subunits can adopt one of three conformations. - L conformation where the B-subunit can loosely bind ADP and Pi - T-conformation where first the B-subunit can tightly bind ADP and Pi and then convert the bound ADP and Pi to ATP - O conformation where the B-subunit can first release ATP and then allow ADP and Pi to enter the active site

Answer 2

For a ring with 10 subunits, 10 protons are required to complete a rotation and one complete rotation yields 3 ATP molecules We can estimate 12 protons are required to complete a cycle so 4 protons are used to produce 1 ATP. NADH translocates 10 protons so it leads to the production of 2.5 ATP molecules. FADH2 translocates 6 protons so it leads to the production of 1.5 ATP molecules.

Answer 3

Components: 7TM Receptor, G-protein complex, Adenylate Cyclase, cAMP, Protein Kinase A. Found in skeletal muscle cells 1) The 7TM receptor binds to a G-protein complex that is "off" when epinephrine or another ligand is not bound 2) When epinephrine binds, the alpha subunit of the G protein releases GDP and GTP binds to it, meaning the GTP-bound a-subunit is on. It then dissociates from the 7TM receptor and the B/y subunits of the G protein also dissociate. 3) When the GTP-bound a-subunit is activated, it binds to adenylate cyclase causes adenylate cyclase to be activated. Adenyl-cyclase can then convert ATP to cyclic AMP or cAMP 4) Elevated levels of cAMP activate Protein Kinase A (PKA) which consist of 4 subunits, 2 regulatory subunits and 2 catalytic subunits 5) When PKA is bound to cAMP and activated, the R-subunits release the C-subunits and the C subunits phosphorylate their target proteins on Ser and Thr 6) Phosphodiesterase then hydrolyzes cAMP to AMP which then allows the R and C subunits to reassociate inactivating PKA 7) The a-subunit of the G protein then hydrolyzes GTP to produce GDP and Pi and the GDP-bound a-subunit can then reassociate with the B and y subunits which then reassociates with the B-adrenergic receptor Epinephrine binds in the skeletal muscle

Answer 4

Components: 7TM Receptor, G-protein complex, Phosphilipase C, diacylglycerol (DAG), inositol-1,4,5-triphosphate (IP3), Protein Kinase C (PKC). Found in liver or smooth muscle cells 1) When epinephrine binds to the 7TM receptor, the alpha subunit of the G protein releases GDP and binds to GTP. The GTP-bound a-subunit then dissociates from the receptor and activates phospholipase C on the membrane 2) Phospholipase C then hydrolyzes phosphotidylinositol-4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3) 3) IP3 causes a rapid release of CA(+2) by opening IP3-regulated calcium ion channels in the endoplasmic reticulum 4) DAG and IP3 then activate PKC which phosphorylates the target proteins that are on Ser and Thr. Calcium is also a partial activator of PKC Epinephrine binds in the liver or smooth muscle

Answer 5

It is secreted by a-cells in the pancreas, binds to the glucagon receptor, and acts via cAMP/PKA which is similar to the B-adrenergic receptor pathway

Answer 6

1) Insulin receptor is phosphorylated upon binding of insulin 2) This phosphorylates the IRS-1 protein 3) This recruits phosphoinositide-3-kinase that adds a phosphate group to the 3 carbon of Phosphatidylinositol 4,5-bisphosphate (PIP2) to form PIP3 4) PIP3 dependent protein kinase (PDK1) then becomes activated and phosphorylates Protein Kinase B (Akt) which phosphorylates the target proteins

Answer 7

1) GLUT1 & GLUT3 are found in almost all mammalian tissue and used for glucose uptake at a constant rate. Active when concentration is above KM, and normal serum glucose is 4-8 mM (KM = 1 mM) 2) GLUT2 is found in liver and pancreatic B cells and found when KM = 15-20 mM 3) GLUT4 is found in muscle and fat cells and used when KM = 5 mM. Insulin stimulates the release of vesicles containing pre-made GLUT4, and endurance training increases the amount of GLUT4 present 4) GLUT5 is found in the small intestine and serves as a fructose transporter

Answer 8

Hexokinase - regulated by negative feedback via Glucose-6-phosphate PFK - regulated by lowering PFK's affinity for fructose-6-phosphate. AMP competes with ATP for allosteric binding so PFK activity is increased when a low ATP/AMP ratio is present, and PFK activity is decreased when a high ATP/AMP ratio is present. Decrease in pH also inhibits PFK Pyruvate kinase - high ATP allosterically inhibits pyruvate kinase and fructose-1,6-bisphosphate activates pyruvate kinase. Unphosphorylated pyruvate kinase also has increased activity, so glucagon also decreases pyruvate kinase activity by phosphorylating pyruvate kinase via the PKA/cAMP pathway. This is because glucagon signals low blood sugar and inhibits glucose metabolism

Answer 9

Glucokinase - primary enzyme that phosphorylates glucose in the liver. Fructose-6-phosphate inhibits glucokinase by lowering the enzyme's affinity for glucose Pyruvate kinase - regulated allosterically by high ATP (which inhibits it) and fructose-1,6-bisphosphate (which activates it)

Answer 10

PFK-1 - regulated allosterically citrate: inhibitor, indicates citric acid cycle is active and glycolysis can slow low pH: inhibitor, acidic metabolites produced by citric acid cycle are present indicating glycolysis can slow down high ATP: inhibits it by lowering the affinity of PFK to fructose-6-phosphate, high ATP indicates glycolysis can slow fructose-2,6-bisphosphate: synthesized from fructose-6-phosphate and activates PFK-1 by increasing PFK's affinity for fructose-6-phosphate and also diminishes the inhibiting effects of ATP PFK-2 - regulated allosterically and through phosphorylation kinase activity: stimulated when PFK-2 is unphosphorylated (inhibited when phosphorylated). When kinase activity is activated fructose-6-phosphate is phosphorylated into fructose-2,6-bisphosphate which activates PFK-1 and stimulates glycolysis activated by inorganic phosphate and inhibited by citrate allosterically. high fructose-6-phosphate levels also stimulate phosphoprotein phosphatase which dephosphorylates PFK-2 and stimulates kinase activity/glycolysis. phosphatase activity: stimulated when PFK-2 is phosphorylated (inhibited when unphosphorylated). When phosphatase activity is activated fructose-2,6-bisphosphate is dephosphorylated into fructose-6-phosphate which inhibits glycolysis fructose-6-phosphate inhibits PFK-2 phosphatase activity allosterically. Also activated by glucagon since PFK-2 is phosphorylated by PKA/cAMP pathway stimulating phosphatase activity/gluconeogenesis.

Answer 11

All of these three enzymes are activated when gluconeogenesis is activated Pyruvate carboxylase: activated by acetyl-CoA and inhibited by ADP Phosphoenol pyruvate carboxykinase: inhibited by ADP Fructose-1,6-bisphosphatase: inhibited by fructose-2,6-bisphosphate and AMP, activated by citrate

Answer 12

E2: high acetyl-CoA concentrations inhibit it and high HS-CoA concentrations activate it (substrate stimulation product inhibition) E3: High NADH/NAD+ ratio inhibits, low NADH/NAD+ ratio activates E1: regulation through phosphorylation occurs where phosphorylated pyruvate dehydrogenase is inactive and unphosphorylated pyruvate dehydrogenase is active pyruvate dehydrogenase kinase: phosphorylates pyruvate dehydrogenase (E1) and renders it inactive, regulated allosterically. Activated by high acetyl-CoA, high NADH/NAD+ ratio, high ATP and inhibited by high ADP and high pyruvate pyruvate dehydrogenase phosphatase: dephosphorylates pyruvate dehydrogenase and renders it active. Activated by high Ca(+2) as a byproduct of the a-adrenergic signal transduction pathway

Answer 13

Allosteric regulation of three enzymes in mammalian cells citrate synthase: inhibited by ATP isocitrate dehydrogenase: activated by high [Ca(2+)] (under stress) and ADP and inhibited by ATP and NADH a-ketoglutarate dehydrogenase: inhibited by ATP, Succinyl-CoA, and NADH In E. Coli isocitrate dehydrogenase is active when unphosphorylated and inactive when phosphorylated and a kinase and phosphatase phosphorylates (deactivates)/dephosphorylates (activates) respectively. isocitrate dehydrogenase kinase is inhibited and phosphatase is activated by the same 5 molecules via allosteric regulation activating TCA: isocitrate, oxaloacetate, pyruvate, 3-phosphoglycerate, phosphoenolpyruvate

Answer 14

UDP-glucose pyrophosphorylase synthesizes UDP-glucose from UTP and Glucose-1-phosphate via the reaction Glucose-1-phosphate + UTP --> UDP-glucose + PPi This reaction is irreversible since PPi spontaneously breaks down into 2Pi immediately in the presence of H2O even though it is thermodynamically reversible. Uridine triphosphate (UTP) is made up of uracil, ribose, and pyrophosphate (3 phosphate groups) and UDP glucose can be formed when terminal phosphates of UTP get removed and initial phosphate can be added to phosphate at 1 C of glucose

Answer 15

Glycogen synthase catalyzes the overall reaction where a glycosyl molecule from UDP-glucose is transferred to the hydroxyl group at C4 of the terminal glucose in a polymer forming a a-1,4-glycosidic bond. It requires a glycogen molecule with a minimum of 4 residues UDP-glucose + Glycogen (n residues) --> UDP + Glycogen (n+1 residues) Glycogenin is an enzyme with it's own substrate (autocatalysis) that is made up of two subunits, and each subunit catalyzes the addition of glycosyl subunit from UDP-glucose to Tyr-194 of another UDP-glucose subunit forming an a-1,4 glycosidic bond. It acts as a primer by polyermizing ~8 glucose molecules and after the primer is formed, glycogen synthase can then further polymerize the glucose molecules into glycogen

Answer 16

When a "string" of at least 11 glycosyl residues are formed, a branching enzyme breaks the chain of glycogen and transfers a string of residues (typically 7) from the non-reducing end to the presumptive branch point and a linkage is formed through an a-1,6-glycosidic bond. A branch is typically seen every 10 residues. Branching allows the number of terminal glucose residues to increase, and since these terminal residues are sites of action for glycogen phosphorylase and synthase, branching increases the rate of glycogen synthesis and degradation.

Answer 17

Glycogen phosphorylase phosphorylates glucose-1-phosphate on the terminal glucose residue (non-reducing end) and protonates the O at the 4 carbon. It uses a phosphate group to break the a-1,4 linkage Glycogen (n residues) + Pi --> glucose-1-phosphate + glycogen (n-1) In order to debranch, glucose residues are removed to the limit dextran by phosphorylase (4th glucose residue from branch glucose) and three enzymes are used Transferase: transfers the 3 terminal glucose residues from outer branch to the center (main and linear branch) a-1,6-glucosidase hydrolyzes the a-1,6-glycosidic bond to release a free glucose molecule Phosphorylase can then continue to remove glucose residues from the non-reducing end producing Glucose-1-phosphate molecules Phosphoglucomutase converts glucose-1-phosphate to glucose-6-phosphate for further metabolism and for export out of the liver to other tissues making glucose-1,6-bisphosphate as an intermediate

Answer 18

In muscle, unphosphorylated glycogen phosphorylase (b form) is inactive and phosphorylated glycogen phosphorylase (a form) is active. Each form has two states where T state is less active (less likely to be phosphorylated) and R state is more active (more likely to be phosphorylated). a-form favors the R-state and b-form favors the T-state

Answer 19

The lowest level of activity is the b form of the T state (1), the second lowest level of activity is the b form of the R state (2), the second highest level of activity is the a form of the T state (3), and the highest level of activity is the a form of the R state (4)

Answer 20

Allosteric regulation only occurs on the b form When ATP is low, AMP levels are high and AMP binds to an allosteric state to stabilize the R-state meaning glycogen phosphorylase activity is elevated When ATP is high, ATP stabilizes the T state making glycogen phosphorylase less active Glucose-6-phosphate stabilizes the T state (less active)

Answer 21

Allosteric regulation only occurs on the a form Glucose mediates the transition of the a-form from the R to the T state reducing glycogen phosphorylase activity

Answer 22

It phosphorylates glycogen phosphorylase at Ser-14 activating it and allowing glycogen to be broken down It contains a y subunit that serves as the catalytic subunit and aBd subunits that serve as regulatory units. The d subunit binds to a Ca(+2) protein called calmodulin, a regulatory protein that activates the enzyme It becomes fully activated in the presence of PKA/cAMP via the B-adregenic receptor (epinephrine) in skeletal muscle, PKA/cAMP via glucagon pathway in the liver, and Ca(+2) via the a-adregenic receptor (epinephrine) in the liver or smooth muscle

Answer 23

Glycogen synthase synthesizes glycogen Unphosphorylated glycogen synthase (a form) is active and phosphorylated glycogen synthase (b form) is inactive PKA phosphorylates glycogen synthase inhibiting it in the muscle and liver, PKB causes it to be activated

Answer 24

Insulin increases the presence of protein kinase B. This phosphorylates glycogen synthase kinase (generally only present in the liver) rendering it inactive. When glycogen synthase kinase is inactive, it cannot phosphorylate glycogen synthase, and since unphosphorylated glycogen synthase (the a form) is active, this means that insulin stimulates glycogen synthesis, and this makes sense since insulin is secreted when blood glucose levels are high

Answer 25

In muscle, protein phosphatatase 1 (PP1) removes the phosphate group of an enzyme regulated by phosphorylation. The GM protein allows PP1 to associate with its target when it is not phosphorylated, and the phosphorylation of the GM protein slows the binding of PP1 with its target. Protein Kinase A phosphorylates the GM protein preventing the binding of an enzyme with PP1 and the removal of a phosphate The phosphorylation of an inhibitor protein by Protein Kinase A also binds to the active site of PP1 and stops it from removing phosphate groups of enzymes PP1 functions to "turn off" both glycogen synthesis and degradation, and epinephrine and glucagon inhibits this process via PKA

Answer 26

The presence of phosphoryated glycogen phosphorylase (a-form) in the R state (low glucose) inhibits PP1 activity and allows glycogen degradation to occur PP1 can dephosphorylate glycogen phosphorylase when in the T state, but in the R state, phosphate groups are hidden after they form a dimer/inhibitory complex When high levels of glucose are present, the T state is stabilized since glucose induces the transition of phosphorylated glycogen phosphorylase (a-form) from R to T state allowing PP1 to become active. When PP1 is active glycogen phosphorylase a can be dephosphorylated (converted to b form) reducing glycogen degradation and glycogen synthase can be dephosphorylated (converted to the active a-form) and stimulating glycogen synthesis

Biochem Exam III Flashcards

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