CMB Exam 2 - Processes Flashcards
How does insulin reverse the action of glucagon?
Via phosphodiesterase-mediated breakdown of cAMP (PKA inactivation) and unregulated enzyme dephosphorylation by protein phosphatases. Also, activation of: PFK2 (and PFK1 indirectly, by synthesis of F-2,6BP), pyruvate kinase, and glycogen synthase. Inactivation of: F-2,6 bisphosphatase, phosphorylase kinase, glycogen phosphorylase, and hormone-sensitive lipase.
Outline the steps of glycolysis, including names of enzymes.
What is the limiting factor of glycolysis? How is this factor replenished?
NAD+ supply is limited for glyceralehyde-3-phosphate dehydrogenase. It must be replenished: in anaerobic conditions by conversion of lactate to pyruvate by lactate dehydrogenase, or in aerobic conditions by a glycerol-3PDH shuttle that passes H2 to the mitochondria.
glycerol-3 phosphate shuttle
In aerobic conditions (esp in brain, skeletal muscle), regenerates NAD+ by passing the hydrogens onto dihydroacetone phosphate (DHAP) forming glycerol-3-phosphate, and then onto flavoproteins in the inner mitochondrial membrane.
malate-aspartate shuttle
Under aerobic conditions (esp in the cardiac muscle, liver and kidney) the hydrogens from glyceraldehyde 3-phosphate are transfered to NAD+ by G3PDH. NAD+ is regenerated by passing the hydrogens on to oxaloacetate forming malate, which enters the mitochondria and participates in the TCA cycle. Aerobic conditions in the cardiac muscle, liver and kidney.
What modulates glycolysis in liver vs muscle/RBCs?
LIVER: pyruvate kinase activates in presence of insulin, glucagon action phosphorylates PFK-2 preventing its product F-2,6-BP from activation PFK-1 (thus inhibiting glycolysis). MUSCLE/RBCs: ATP/(AMP+ADP) ratio; ATP binds to and inhibits PFK-1 and pyruvate kinase, AMP activates PFK-2.
How does glycogen synthesis occur?
Glucose –(glucokinase)–> Glu6P –(phosphoglucomutase)–> Glu1P + UTP —-> UDP-glucose + glycogenin –(glycogen synthase)–> elongated glycogen –(branching enzyme)–> branched/elongated glycogen
How does glycogen degradation occur?
Under the influence of glucagon, glycogen phosphorylase uses a phosphate (instead of water) to split glucose off glycogen, leaving Glu1P. WHEN THERE ARE 4 LEFT IN A BRANCH (ie “limit dextrin”): transferase moves 3 over to the straight chain, and an α-1,6-glucosidase removes the last 1. The free Glu1P is changed to Glu6P by phosphoglucomutase and then to glucose by Glu-6-phosphatase (expressed ONLY in the liver).
Outline the mechanism by which glucagon/epinephrine cause glycogen degredation.
Both glucagon and epinephrin activate adenylyl cyclase, which turns ATP to cAMP. cAMP causes the dissociation of the regulatory subunits of PKA from it’s catalytic subunits. PKA activates phosphorylase kinase, which activates phosphorylase. Phosphorylase uses a phosphate to separate Glu1P from the glycogen chain. PKA also inactivates glycogen synthase to avoid cycling.
What role does fat (fatty acids) have in gluconeogenesis?
Glucagon signals release of FFAs from adipose tissue (by hormone-sensitive lipase), which is broken down in the liver and provides ATP for gluconeogenesis.
The Cori Cycle
The recycling by the liver of the lactate produced in RBCs; lactate becomes the principle substrate for gluconeogenesis.
Walk through all the steps of gluconeogenesis, starting with lactate.
Lactate + NAD+ –(lactate dehydrogenase)–> pyruvate + NADH –(enters mitochondria)–> pyruvate –(pyruvate carboxylase, biotin)–> OAA –(transamination)–> Asp –(leaves mitochondria)–> Asp –(deamination)–> OAA –(phosphoenol-pyruvate carboxylase)–> PEP –(enolase)–> 2-phosphoglycerate –(phosphoglycerate transmutase)–> 3-phosphoglycerate –(phosphoglycerate kinase)–> 1,3-bisphosphoglycerate + NADH –(glyceraldehyde-3-phosphate dehydrogenase)–> glyceraldehyde-3-phosphate + NAD+ –(aldolase)–> fructose-1,6-bisphosphate –(fructose-1,6-bisphosphatase in the absence of F-2,6-P)–> fructose-6-phosphate –(phosphohexose isomerase)–> glucose-6-phosphate –(enters ER)–> glucose-6-phosphate –(glucose-6-phosphatase)–> GLUCOSE! :D take it to the blood!
Describe the process of phosphoenol pyruvate formation in the absence of lactate.
First we need to generate pyruvate (since liver doesn’t do glycolysis in fed state); in the absence of lactate, amino acids like alanine (but not leucine or lysine) are turned into pyruvate. Pyruvate enters mitochondria and is carboxylated to oxaloacetate (pyruvate carboxylase, biotin dependent). OAA is reduced to malate, leaves the mitochondria and is re-oxidized to OAA. OAA is then decarboxylated (phosphoenol-pyruvate carboxylase, GTP) to form PEP.
Outline the oxidative pentose phosphate pathway.
Glucose-6-phosphate + NADP+ –(glucose-6-phosphate dehydrogenase)–> NADPH+ a lactone structure that is acted on by another enzyme and NADP+ —-> NADPH + CO2 + ribulose-5-phosphate (NOT RIBOSE-5-phosphate). So, a six carbon sugar has been oxidized to 2 NADPH and a 5 carbon sugar.
Explain how macrophages generate bacteriocidal ROS.
MAcrophages take the NADPH from the PPP: NADPH + O2 –(NADPH oxidase)–> O2- + NADP+ –(superoxide dismutase)–> H2O2, which can be dumped on bacteria or –(myeloperoxidase)–> HOCl.
Outline the conversion of pyruvate to acetyl-CoA.
1) E1*TPP displaces CO2 from pyruvate and donates an H+. 2) The hydroxyethyl group is transfered to oxidized (S-S) lipoyllisine on E2, reducing the lipoyllysine and converting the hydroxyethyl to an acetyl group. 3) E2 then attaches CoA-SH to the acyl lipoyllysine forming acetyl-CoA, which leaves. 4) Lipoyllysine is still reduced, so E3 uses FAD to oxidize it. 5) NAD+ then oxidizes the FADH2.
Describe the regulation of PDH
ACTIVATED: allosterically via AMP, CoA and NAD+, Ca++ (ie low ATP + nececssary substrates) and covalently via dephosphorylation. INHIBITION: allosterically via ATP, acetyl-CoA, and NADH, and covalently via autophosphorylation of E1.
Outline the “important” steps of TCA (according to Dory’s slides).
You add acetyl-CoA (2 carbons) to OAA and make citrate. [In the liver, citrate leaves for the cytoplasm and is reconverted to acetyl-CoA]. The other important things to remember are that the first CO2 comes off via isocitrate dehydrogenase (yielding 1 NADH), the next comes of via α-ketoglutarate dehydrogenase (analogous to PDH; yields 1 NADH), then there’s substrate-level phosphorylation producing GTP and succinate. Succinate dehyrogenase (a member of the e-transport chain) uses FAD to oxidize succinate, then fumarate is converted to malate which is oxidized by malate dehydrogenase (yields 1 NADH) to OAA. So: we add 2 carbons to the cycle and we get 3 NADH, 1 FADH2, and some substrate-level phosphorylation.
How does alcohol inhibit gluconeogenesis?
Cyctosolic alcohol dehydrogenase turns all the NAD to NADH, which always drives pyruvate to lactate, inhibiting gluconeogenesis. (alcohol also enters the mitochondria and turns into acetaldehyde and the acetate in a process that uses up mitochondrial NAD as well).
Outline the general steps of the e- transport system.
NADH dehydrogenase in Complex I oxidizes NADH to NAD+, simultaneously pumping an H+ across the membrane. Complex I (from NADH from wherever), Complex II (from FADH2 from TCA), Glycerol 3-phosphate dehydrogenase (from FADH2 glycerol 3-phosphate shuttle in glycolysis), and ETF (from β-oxidation) dump e-s onto CoQ. CoQ shuttles e-s to Complex III (another H+ pump) then to cytochrome C then to Complex IV (another pump; this is where O2 is needed + H+ —-> H20). The H+ ion gradient drives Complex V (ATP synthase).
How are ROS generated in the mitochondria? How do mitochondria handle the ROS?
In hypoxic conditions the transfer of e-s slows down enough that O2 can react with either Complex I or III to form superoxide. O2- –(superoxide dismutase)–> H2O2 –(glutathione peroxidase)–> H2O. Glutathione peroxidase also leaves glutathione dimers in the oxidized state, and NADPH regenerates them.
How do we get Pi and ADP into the mitochondrial matrix to make ATP?
Pi enters using H+ symport (thanks to the pumps). ADP enters using ATP/ADP antiport.
Outline the process of lypolysis.
Perilipin is phosphorylated by PKA causing it to open up and give HSL access to the triacylglycerol inside. HSL removes the first FFA from TG, other lipases release the other 2.
Outline the steps of the carnitine cycle.
The FA is brought into the intermembrane space where it is converted to Acyl-CoA. Acyl-CoA + carnitine –(carnitine-palmitoyl acyltransferase 1)–> CoA + acyl-carnitine. Acyl-carnitine can cross the other lipid layer and is reconverted to acyl-CoA by CPT2, and it can then undergo β-oxidation.
Outline the steps of β-oxidation.
Acyl-CoA dehydrogenase reduces FAD to put a double bond in the β position on the acyl group (ie CoA-S-CO-αC-βC). Water is added accross the double bond, adding a hydroxyl group to the βC. Next, another dehydrogenase uses NAD+ to oxidize the hydroxyl to a ketone group. Finally, a thiolase attacks the ketone and knocks off acetyl-CoA, reducing the length of the FA chain by 2C.
Outline the net reaction of the synthesis of palmitic acid from acetyl-CoA.
8 Acetyl-CoA + 14 NADPH + 14 H+ + 7 ATP —-> Palmitate + 14 NADP+ + 7 ADP + 7 Pi + 8 CoA-SH + 6 H2O. Overall, fatty acid synthesis is a reductive process that requires energy.
Outline the generation of Acetyl-CoA and NADPH for fatty acid synthesis.
Glucose –(glycolysis)–> pyruvate –(enters mitochondrion)–> pyruvate –(pyruvate carboxylase and dehydrogenase)–> OAA and acetyl-CoA —-> citrate –(leaves mitochondrion)–> citrate + ATP –(citrate lyase)–> OAA + acetyl CoA. Acetyl-CoA is used for FA synthesis, OAA gets recycled in a process that generates NADPH (OAA + NADH –(cytosolic malate dehydrogenase)–> NAD+ + malate; malate + NADP+ –(malic enzyme)–> pyruvate + NADPH + CO2). The pentose phosphate pathway also produces NADPH.
Summarize fatty acid synthesis from acetyl-CoA.
Acetyl-CoA –(acetyl-CoA carboxylase + biotin)–> malonyl-CoA. With the help of the ACP carrier protein and the reductive hep of NADPH, FA synthase joins the malonyl-CoA molecules together, extending FAs 2 carbons at a time until the 16 carbon palmitoyl-CoA is achieved. Further elongation or desaturation occurs in the ER.
Explain the function and regulation of acetyl-CoA carboxylase.
A biotin-dependent enzyme (carboxylase) that converts acetyl-CoA to malonyl-CoA during FA synthesis. Glucagon inactivates via phosphorylation (and degradation of the large enzyme polymers), insulin activates via dephosphorylation. Citrate (present when insulin is present) activates it by facilitating it’s polymerization, while its product malonyl-CoA inhibits it.
Briefly outline the synthesis of purines. How many ATP are utilized?
First, Ribose-5-phosphate + ATP –(PRPP synthetase)–> PRPP +AMP. Then PRPP + glutamine –(PRPP amidotransferase)–> phosphoribosylamine + glutamate. Phosphoribosylamine undergoes 7 more modifications to form inosine monophosphate. 4 ATP are used up in this process.
How are NMPs converted to NDPs?
Adenylate kinase or guanylate kinase phosphorylate them, BOTH using ATP.
Where can purine synthesis be regulated? (3 places)
PRPP synthetase and PRPP amidotransferase can both be inhibited by purine nucleotides (product inhibition). Also, reactions from IMP to NMPs (ie adenylosuccinate synthetase and IMP dehydrogenase) are inhibited by NMPs.
Outline purine degradation.
AMP needs to be deaminated (by either AMP deaminase or adenosine deaminase) and dephosphorylated (by 5’-nucleotidase) to inosine. Purine nucleoside phosphorylase uses a Pi to cleave inosine into ribose-1-phosphate and hypoxanthine, after which xanthine oxidase uses FAD to oxidize hypoxanthine to xanthine and then again to uric acid. Guanine on the other hand, does not need to be deaminated, just dephosphorylated (5’-nucleotidase) and cleaved from the ribose (purine nucleoside phosphorylase) to be freed, and then guanine deaminase turns it straight into xanthine, which is oxidized by xanthine oxidase to uric acid.
How do we salvage dietary and turnover purines?
Adenine phosphoribosyltransferase (APRT) and Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) turn purine bases into nucleotides. Both use PRPP as the source of the ribose-5-phosphate group.
Outline the steps of pyrimidine synthesis from glutamine to CTP.
Glutamine + {CO2 + 2 ATP} –(carbamoyl phosphate synthetase II)–> {glutamate + 2 ADP + Pi} + carbamoyl phosphate + aspartate –(aspartate transcarbamoylase)–> N-carbamoyl-aspartate –(dihydroorotase)–> L-dihydroorotate + NAD+ –(dihydroorotate dehydrogenase)–> {NADH} + orotate + PRPP –(orotate phosphoriboyltransferase)–> oritidine monophosphate –(oritidylate decarboxylase)–> uridine monophosphate —-> UDP —-> UTP + {glutamine + ATP} –(CTP synthetase)–> {glutamate + ADP + Pi} + CTP!!
Outline the general steps of pyrimidine degradation.
Deamination (only in the case of cytidine to uridine); nucleoside phosphorylase cleavage of the sugar (turning uridine to uracil or 2-deoxythymidine to thymine); reduction by NADPH; ring cleavage by water; hydrolysis by water to release CO2 and NH3. The products (β-alanine and β-aminoisobutyrate) are excreted in the urine.
Explain ribonucleotide reductase structure/regulation.
Ribonucleotide reductase has specificity sites (whatever is bound increases production of itself?), activity sites (to regulate; ATP activates, dATP inhibits), and reduction sites (where the NDP is reduced to dNDP). Is inhibited by hydroxyurea
Outline the process of ubiquitination.
Ubiquitin –(E1 + ATP)–> ubiquitin-S-E1 –(E2)–> ubiquitin-S-E2 + target protein-Lys-NH2 –(E3)–> target-Lys-NH-ubiquitin.
Outline protein degradation in the stomach.
Gastrin is produced by stomach G cells in response to protein-rich meal; activates chief cells (secrete pepsinogen) and parietal cells (secrete HCl which denatures proteins and also activates pepsinogen autocatalytic cleavage); allowing pepsin to start to cleave proteins (ie formation of peptone).
Outline protein degradation in the small intestine.
The low pH triggers pancreatic release of secretin (stimulates the pancreas to secrete bicarbonate) and cholecystokinin (stimulates release of trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidases A & B). Enteropeptidase activates trypsin, trypsin activates the other zymogens.
How are AAs pumped from the intestinal lumen to the serosal side of the epithelium?
Na+ symport (gradient maintained with an NA/K pump) into the epithelial cell, then facilitated transport out.
Outline the flow of nitrogen from amino acids to ammonia and urea.
Amino acids pass their NH3 to pyruvate forming alanine via transaminases. Ala passes the NH3 to α-ketoglutarate via alanine transaminase, forming glutamate. Glutamate can either give off ammonium (via glutamate dehydrogenase) or can pass the NH3 to OAA forming aspartate, which is processed into urea.
Track the path of ammonia in the glucose-alanine cycle.
In muscles, NH3 + α-ketoglutarate –(glutamate dehydrogenase)–> glutamate; NH3 is passed to pyruvate (alanine aminotransferase) forming alanine; alanine is sent to the liver; NH3 passed back to α-ketoglutarate (alanine aminotransferase) forming {pyruate} and glutamate –(glutamate dehydrogenase)–> NH3 is released, then converted to urea.
Outline the urea cycle, starting with NH4+ in the mitochondrion.
