CMB Exam 2 - Processes

Question 1

How does insulin reverse the action of glucagon?

Answer 1

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.

Question 2

Outline the steps of glycolysis, including names of enzymes.

Answer 2

Question 3

What is the limiting factor of glycolysis? How is this factor replenished?

Answer 3

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.

Question 4

glycerol-3 phosphate shuttle

Answer 4

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.

Question 5

malate-aspartate shuttle

Answer 5

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.

Question 6

What modulates glycolysis in liver vs muscle/RBCs?

Answer 6

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.

Question 7

How does glycogen synthesis occur?

Answer 7

Glucose –(glucokinase)–> Glu6P –(phosphoglucomutase)–> Glu1P + UTP —-> UDP-glucose + glycogenin –(glycogen synthase)–> elongated glycogen –(branching enzyme)–> branched/elongated glycogen

Question 8

How does glycogen degradation occur?

Answer 8

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).

Question 9

Outline the mechanism by which glucagon/epinephrine cause glycogen degredation.

Answer 9

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.

Question 10

What role does fat (fatty acids) have in gluconeogenesis?

Answer 10

Glucagon signals release of FFAs from adipose tissue (by hormone-sensitive lipase), which is broken down in the liver and provides ATP for gluconeogenesis.

Question 11

The Cori Cycle

Answer 11

The recycling by the liver of the lactate produced in RBCs; lactate becomes the principle substrate for gluconeogenesis.

Question 12

Walk through all the steps of gluconeogenesis, starting with lactate.

Answer 12

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!

Question 13

Describe the process of phosphoenol pyruvate formation in the absence of lactate.

Answer 13

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.

Question 14

Outline the oxidative pentose phosphate pathway.

Answer 14

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.

Question 15

Explain how macrophages generate bacteriocidal ROS.

Answer 15

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.

Question 16

Outline the conversion of pyruvate to acetyl-CoA.

Answer 16

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.

Question 17

Describe the regulation of PDH

Answer 17

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.

Question 18

Outline the “important” steps of TCA (according to Dory’s slides).

Answer 18

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.

Question 19

How does alcohol inhibit gluconeogenesis?

Answer 19

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).

Question 20

Outline the general steps of the e- transport system.

Answer 20

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).

Question 21

How are ROS generated in the mitochondria? How do mitochondria handle the ROS?

Answer 21

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.

Question 22

How do we get Pi and ADP into the mitochondrial matrix to make ATP?

Answer 22

Pi enters using H+ symport (thanks to the pumps). ADP enters using ATP/ADP antiport.

Question 23

Outline the process of lypolysis.

Answer 23

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.

Question 24

Outline the steps of the carnitine cycle.

Answer 24

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.

Question 25

Outline the steps of β-oxidation.

Answer 25

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.

Question 26

Outline the net reaction of the synthesis of palmitic acid from acetyl-CoA.

Answer 26

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.

Question 27

Outline the generation of Acetyl-CoA and NADPH for fatty acid synthesis.

Answer 27

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.

Question 28

Summarize fatty acid synthesis from acetyl-CoA.

Answer 28

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.

Question 29

Explain the function and regulation of acetyl-CoA carboxylase.

Answer 29

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.

Question 30

Briefly outline the synthesis of purines. How many ATP are utilized?

Answer 30

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.

Question 31

How are NMPs converted to NDPs?

Answer 31

Adenylate kinase or guanylate kinase phosphorylate them, BOTH using ATP.

Question 32

Where can purine synthesis be regulated? (3 places)

Answer 32

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.

Question 33

Outline purine degradation.

Answer 33

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.

Question 34

How do we salvage dietary and turnover purines?

Answer 34

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.

Question 35

Outline the steps of pyrimidine synthesis from glutamine to CTP.

Answer 35

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!!

Question 36

Outline the general steps of pyrimidine degradation.

Answer 36

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.

Question 37

Explain ribonucleotide reductase structure/regulation.

Answer 37

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

Question 38

Outline the process of ubiquitination.

Answer 38

Ubiquitin –(E1 + ATP)–> ubiquitin-S-E1 –(E2)–> ubiquitin-S-E2 + target protein-Lys-NH2 –(E3)–> target-Lys-NH-ubiquitin.

Question 39

Outline protein degradation in the stomach.

Answer 39

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).

Question 40

Outline protein degradation in the small intestine.

Answer 40

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.

Question 41

How are AAs pumped from the intestinal lumen to the serosal side of the epithelium?

Answer 41

Na+ symport (gradient maintained with an NA/K pump) into the epithelial cell, then facilitated transport out.

Question 42

Outline the flow of nitrogen from amino acids to ammonia and urea.

Answer 42

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.

Question 43

Track the path of ammonia in the glucose-alanine cycle.

Answer 43

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.

Question 44

Outline the urea cycle, starting with NH4+ in the mitochondrion.

Answer 44