module 2 lecture
PRK-1 is regulated by allosteric effectors
1. AMP and ATP 2. citrate 3. fructose 2, 6-biphosphate (regulated by glucagon and epinephrine)
described hormonal control (for gluconeogenesis)
1. Glucagon and insulin regulate the activity of enzymes through covalent modulation. ---Glucagon decreases the concentration of Fructose 2,6-biPO4, which inhibits Glycolysis and stimulates Gluconeogenesis, in the liver by stimulating the phosphorylation of the bifunctional enzyme "PFK-2/Fructose 2,6 biphosphatase". ---Glucagon stimulates Gluconeogenesis and inhibits Glycolysis. 2. Glucagon and Insulin also have long-term effects on concentration of hepatic enzymes by induction or repression of enzyme synthesis. ---Glucagon increases synthesis of PEPCK and glucose 6-phosphatase and represses synthesis of glucokinase and pyruvate kinase. --Glucagon stimulates Gluconeogenesis and inhibits Glycolysis.
describe the TCA cycle
1. Products of carbohydrates, fat and amino acids feed into this cycle and are totally oxidized to CO2 and H2O with generation of large amount of ATP. 2. Occurs in mitochondria 3. Some intermediates can be converted to glucose (gluconeogenesis) and some can be converted to certain amino acids by transamination.
reactions of the TCA cycle (that produce energy)
1. Reaction 3: Isocitrate Dehydrogenase converts isocitrate to alpha-ketoglutarate by dehydrogenation and decarboxylation. A molecule of CO2 is removed and NAD is reduced to NADH (*first NADH in the cycle produced*) 2. Reaction 4: Alpha-Ketoglutarate Dehydrogenase converts ketoglutarate to succinyl CoA by decarboxylation/dehydrogenation A molecule of CO2 and NADH is produced (second NADH in the cycle produced) Alpha-ketoglutarate dehydrogenase is a multienzyme/cofactor system similar to pyruvate dehydrogenase The product of this reaction, Succinyl CoA is an energy rich thiolester (similar to acetyl CoA) 3. Reaction 5: Succinyl CoA synthase The energy rich thiol ester produces a GTP by "substrate level phosphorylation" 4. Reaction 6: Succinate Dehydrogenase coverts Succinate to Fumarate FADH2 is produced. 5. Reaction 8: Malate Dehydrogenase converts malate to oxaloacetate. Completes the cycle and regenerates oxaloacetate. Reduces NAD to NADH (third NADH in the cycle produced)
regulation of gluconeogenesis
1. allosteric regulation 2. hormonal regulation
2 types of regulation of pyruvate dehydrogenase
1. allosteric regulation (feedback inhibition) --products of the reaction (*acetyl CoA and NADH*) inhibit the enzyme 2. covalent modulation --a form: active, dephosphorylated --b form: inactive, phosphorylated
what are the regulatory enzymes of the TCA cycle
1. citrate synthase: negatively regulated by ATP but the primary regulation is availability of the substrate 2. isocitrate dehydrogenase: negatively modulated by ATP and NADH and stimulated by ADP and AMP 3. alpha-ketoglutarate dehydrogenase: ATP, GTP, NADH, and succinylcholine CoA (product) inhibit this enzyme complex
what are pyruvate formed from?
1. glucose and other monosaccharides (glycolysis in cytoplasm, under aerobic conditions) 2. AA: alanine, serine, cysteine 3. glycerol (minor)
enzymes that "go around" the irreversible steps of glycolysis that are involved in the regulation of gluconeogenesis
1. pyruvate carboxylase 2. PEP carboxykinase (PEPCK) 3. fructose 1,6 bisphosphotase 4. glucose 6 phosphatase
what is the Km for glucokinase?
10 Km
how many ATP are produced for one acetyl CoA in the Krebs cycle?
9 ATP from 3 NADHs, 2 ATP from 1 FADH and 1 GTP total: 12
describe electron transport chain
A series of protein complexes located at the the inner membrane of the mitochondria. function as electron carriers. Electrons released from NADH and FADH2 are passed from one electron carrier to another and are finally combined with oxygen, the final electron acceptor.
isocitrate dehydrogenase is negatively affected by ______ and _________
ATP and NADH **plenty of energy in the cell, no more glycolysis
how does AMP and ATP regulate enzyme PFK-1
ATP inhibits PFK-1 while AMP stimulates its activity PFK-1 is inhibited at concentrations of ATP normally present in cells the levels of AMP automatically go up in a cell when ATP levels decrease so AMP is a signal of energy status of the cell
sources of acetyl CoA
CHO (glycogen), triglycerides (FFA) and protein (AA) catabolism produce acetyl CoA
described allosteric regulation (for gluconeogenesis)
Fattyacid oxidation promotes gluconeogenesis and inhibits glycolysis by: 1. An increase in concentration of mitochondrial acetyl CoA, which is a positive regulator of pyruvate carboxylase. 2. The increased concentration of acetyl CoA leads to the synthesis of citrate. Citrate is a negative effector of the enzyme PFK-1 and thus results in a decreased production of Fructose 1,6-biPO4, which is an activator of the enzyme pyruvate kinase. So, pyruvate kinase is also inhibited. 3. Increased fatty acid oxidation produces more ATP, which leads to a decrease in AMP. This will favor gluconeogenesis and inhibit glycolysis.
anaplerotic reactions of the TCA cycle
In the TCA cycle, oxaloacetate is regenerated during the cycle However, intermediates of the cycle are removed for biosynthetic pathways Hence, for cycle to remain functional, a source is required to replenish the loss of oxaloacetate. The reactions that supply 4 or 5 carbon intermediates to the cycle are called "anaplerotic" reactions (mostly amino acids). *The most important reaction is catalyzed by pyruvate carboxylase, converts pyruvate to oxaloacetate*. **keep the kerb cycle going**
what inactivated pyruvate kinase
Inactivated by phosphorylation (covalent modulation). 1. Glucagon increases the level of cAMP which activates protein kinase. 2. Protein kinase phosphorylates pyruvate kinase, making it inactive (b form).
regulation of the enzyme PFK-1
Most cells can use Glucose 6-PO4 for Glycogen synthesis or pentose phosphate pathway, so hexokinase/glucokinase is not the first enzyme "unique" to glycolysis. The Glucose 6-PO4 isomerase catalyzed reaction is reversible, hence not regulated. Therefore, PFK-1 catalyzes the first unique step of glycolytic pathway, which commits the cell to metabolism of glucose by glycolysis.
what happens when fluctose 2,6 biPO4 is increased
PFK -1 is increased and glycolysis is increased
regulation of pyruvate dehydrogenase
The enzyme *pyruvate dehydrogenase kinase* phosphorylates pyruvate dehydrogenase and "inactivates" it -pyruvate dehydrogenase kinase is regulated by allosteric modulation: ---stimulated by Acetyl CoA and NADH (products of the pyruvate dehydrogenase reaction) and ATP ---inhibited by pyruvate (substrates of the pyruvate dehydrogenase reaction) The enzyme *pyruvate dehydrogenase phosphatase dephosphorylates* and "activates" pyruvate dehydrogenase -In skeletal muscle, release of Ca++ during contraction will activate phosphatase and stimulate oxidation of pyruvate and energy production
what is the most important anaplerotic reaction?
The most important reaction is catalyzed by pyruvate carboxylase, converts pyruvate to oxaloacetate
what happens when blood glucose is high (Bifunctional Enzyme PFK-2/F 2,6-biphosphatase)
The role of insulin in regulation of fructose 2,6-biPO4 levels is not quite clear Insulin opposes the effect of glucagon. Also, insulin lowers cAMP levels, thus inhibiting protein kinase and activating phosphoprotein phosphatase. Insulin therefore stimulates glycolysis
is the reaction from citrate to isocitrate reversible?
YES
is citrate a postive regulator of FA synthesis?
YES (major one)
what happens with citrate levels are high in the cytoplasm?
activates FA synthesis **if making energy from FA, dont need glucose **negatively regulate PFK-1
a ________________ enzymes carries out both the synthesis and degradation of fructose 2,6 bisphosphate
bifunctional enzymes ex: phosphofructokinase 2/fructose 2,6 biphosphatase **has kinase activity and phosphatase activity
how is fructose 2,6 bisphosphate destroyed
destroyed by being converted to fructose 6-phosphoate by fructose 2,6 biphosphatase **removal of phosphate
what happens with fructose 2, 6 biphsphate levels are high?
encouraging glycolysis inhibiting gluconeogenesis
what stimulates glycolysis IN THE HEART
epinephrine **as a part of a mechanism to meet the increased demand for ATP caused by epinephrine-signaled increase in work load
what inhibits glycolysis IN THE LIVER
epinephrine **in the liver to conserve glucose for use by other tissue
what activates pyruvate kinase
fructose 1,6 bosphosphate 1. Regulation of pyruvate kinase is linked with the activity of enzyme PFK-1. 2. If PFK-1 is active, then concentration of Fructose 1,6-biPO4 increases which in turn activates Pyruvate Kinase.
what activates glucokinase?
fructose 1-PO4 **could inhibit it too
how does fructose 2, 6 bisphosphate regulate enzyme PFK-1
fructose 2, 6 bisphosphate is a positive allosteric regulator of PFK1 and negative regulator of fructose 1,6 biphosphotase
what positivity effects PFK-1 regulation?
fructose 2,6 biphosphate high levels of AMP
where is fructose 2, 6 bophosphate synthesized from?
fructose 6 phosphate **early step in glycolysis
what inhibits glucokinase?
fructose 6-PO4 **next step in glycolysis
what controls bifunctional enzyme PFK-2/F 2,6-bisphosphatase
glucagon (from pancreas) and epinephrine (from pancreas)
regulations of glucokinase
glucokinase (expressed in LIVER cells) glucokinase has a much higher Km for glucose (~7 mM) than hexokinase --> LOW affinity for glucose it is NOT inhibited by glucose 6-PO4, but is inhibited by *fructose 6-Po4* and is activated by *fructose1 1-PO4* in is an inducible enzyme (concentration can be increased)
____________ has a different isozyme of the bifunctional enzyme
heart **phosphorylation by protein kinase occurs at a different site that activates rather than inhibits PFK 2 and *produces an increase rather than decrease in fructose 2,6 biPO4 levels*
how does high Km of glucokinase
helps the liver to "buffer" blood glucose levels --> low affinity for glucose, allows the liver to not use up the glucose in the blood and allow the peripheral tissue get the glucose glucose equilibrates acress the liver plasma membrane and concentration is equal to that of BG (~5mM) since Km for glucokinase for glucose is very high (~7mM), the liver doesn't use much glucose at normal BG concentration any increase in blood glucose concentration leads to a proportional increase in the rate conversion of glucose --> glucose 6 PO4 in the liver any decrease in the concentration of glucose results in a decrease in the rate of phosphorylation of glucose in the liver *so the liver uses glucose at a significant rate only when BG levels are very high to buffer BG level*
regulations of hexokinase
hexokinase (expressed in all tissues except LIVER) hexokinase found in most tissues, has a low Km for glucose (<0.1 mM) relative to normal blood concentration of glucose (~5mM) --> meaning has a HIGH affinity for glucose hexokinase is strongly inhibited by glucose 6-PO4 (product of reaction) --feedback inhibition
what the 3 irreversible reactions in glycolysis
hexokinase/glucokinase (both do the same thing of phosphylate glucose when it enters the cell) phosphofructokinase 1 (PFK 1; rate limiting enzyme) pyruvate kinase **these enzymes are regulated by allosteric regulation and/or covalent modifications
what negativity effect PFK-1 regulation
high levels of ATP high levels of citrate
what the bifunctional enzyme is phosphorlyted what happens?
inactivates the phosphofructo-2 site so kinase activity is reduced reduce the synthesis of fructose 2, 6 phosphate active phosphorylation--> active the fructose 2, 6 biphosphatase site
how does intracellular citrate regulate enzyme PFK-1
increase citrate concentration in the cytosol as a result of Krebs cycle backing up signals that energy levels are adequate and inhibits PFK-1 resulting in decreased glycolysis
when the bifunctional enzymes is dephosphorlyated what happens?
kinase activity is active
describe mechanism of oxidation of pyruvate to acetyl CoA
linking reaction of glycolysis and TCA *irreversible reaction* enzyme pyruvate dehydrogenase --an emzyme complex found only in *mitochondria* converts pyruvate to acetyl CoA --consists of several cofactors, coenzymes and enzymes
what b vitamins are important in TCA cycle?
niacin (NAD) thiamine riboflavin (FAD)
regulation of bifunctional enzyme PFK-2/F 2,6-bisphosphatase
phosphorylation of the bifunctional enzyme causes "inactivation" of the PFK-2 site (synthesis of F 2,6 bisphosphate) and "activation" of F 2,6-biphosphatase site (hydrolysis of F 2,6 bisphosphate) dephosphorylation of this bifunctional enzyme has an opposite effect 1. When blood glucose is low, Glucagon is produced and stimulates the cAMP production in the liver 2. cAMP stimulates protein kinase, which phosphorylates the bifunctional enzyme. That causes "inactivation" of the PFK-2 site and "activation" of F 2,6- biphosphatase site, thus decreasing Fructose 2,6-biphosphate. 3. A decrease in Fructose 2,6-biphosphate makes PFK-1 less effective but makes fructose 1,6-biphosphatase more effective Inhibits glycolysis but stimulates gluconeogenesis Blood glucose rises
how is fructose 2, 6 bisphosphate produced
produced from fructose 6-phosphate by phosphofructokinase 2
what are some metabolic fates of pyruvate
pyruvate is metabolized depending on the tissue and the metabolic state of the tissue transamination --> alanine carboxylation --> PAA reduction --> lactate (anaerobic) oxidative decarboxylation --> acetyl CoA (aerobic) --> TCA
source sources and fates of succinylcholine CoA
succinylcholine CoA maybe formed either from: 1. alpha-ketoglutarate in the TCA cycle OR 2. from methylmalonyl COA from the breakdown of odd C chain fatty acids OR 3. the branched chain amino acid valine and isoleucine succinylcholine COA can be converted to succinate in the TCA cycle or can leave to be used for *porphyrin synthesis*
how is glucokinase is an "inducible" enzyme
the amount of glucokinase can be increased or decreased controlled by hormones: increase in BG --> increased in insulin --> *insulin* stimulates the transcription of glycokinase gene and thus increases the amount of glucokinase the absence of insulin, makes the dietetic patient deficient in glucokinase, in spite of high BG levels (NOT producing own insulin) **insulin major driver of expression for glucokinase
regulation of the TCA cycle
the supply of acetyl CoA, whether from pyruvate or FA is crucial in determining the rate of TCA cycle inhibitory agent or metabolic condition that interrupts the supply of O2, the continuous supply of ADP, or the sources of reducing equivalents would shut down the cycle
oxidative phosphorylation
the synthesis of ATP linked to the oxidation of NADH and FADH2 and transport of electron through ETC 1 NADH oxidized in the ETC leads to synthesis of 2.5 ATP 1 FADH2 oxidized in the ETC leads to synthesis of 1.5 ATP
in the liver: fructose --> fructose 1-PO4
this may be related to an adverse effect associated with excessive dietary fructose consumption --> thus inhibiting glucosecokinase e.g. increased hepatic CHO utilization, lipogenesis, and hypertriglycerdimia
T/F: Pyruvate kinase is also induced in the liver by a combination of high CHO-high insulin levels.
true
what is the Km for hexokinase?
~1 Km
