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1

Hexokinase and glucokinase are involved in what important reaction?

At the beginning of glycolysis, priming glucose. IRREVERSIBLE REACTION
* D-glucose --> Glucose-6-phosphate

2

What does the function of hexokinase or glucokinase result in?

*these are tissue specific glucose primer reactions (IRREVERSIBLE reactions)
*net negative of charge on glucose traps glucose in cell
*conservation of energy (mostly) as ATP is expended to make more ATP by making glucose available for glycolysis
*next enzyme in glycolysis pathway needs the phosphate to bind selectively

3

what are the important differences between hexokinase and glucokinase?

hexokinase
*present in all cells
*low km and super specific for sugars, but NOT specific for glucose
*inhibited by glucose-6-phosphate
glucokinase
*super specific for glucose
*liver and pancreatic beta-cells
*high km for glucose
*inhibited by fructose-6-P

4

what is NORMAL blood glucose concentration?

4-5mM
*multiply by 18 to get mg/dL
*72-90 md/dL

5

What reaction concerning fructose is super important to know (glycolysis)?

The SECOND ATP investment in the glycolysis pathway
*Fructose-6-P + ATP --> Fructose-1,6-bis-P + ADP
*enzyme = phosphofructokinase I

6

Phosphofructokinase I is important for what reaction?

The SECOND ATP investment in the glycolysis pathway
*Fructose-6-P + ATP --> Fructose-1,6-bis-P + ADP

7

what is the reaction that COMMITS the cell to glycolysis pathway?

*the IRREVERSIBLE ATP-investment in glycolysis of:
*Fructose-6-P + ATP --> Fructose-1,6-bis-P + ADP
*catalyzed by PFK1 = phosphofructokinase 1

8

What stimulates and inhibits PFK1?

*PFK1 = phosphofructokinase 1
*AMP = stimulates
*Fructose-2,6-bis-P is the MOST POTENT stimulator
*F26BP is made by PFK2
*ATP inhibits

9

how does cAMP play a role in PFK1 activity?

When cAMP is LOW (high insulin/glucagon ratio, or in a fed state) there is a preferential de-phosph of PFK2,
*de-phosph PFK2 is ACTIVE
*MORE F26BP generation
*since F26BP is the most potent stimulator of PFK1 then low cAMP is indirectly a reason for HIGH PFK1 activity
*this makes sense as cAMP is low when insulin is high and glucagon low, or when there is a need for glycolysis

10

PFK1 is pretty much regulated by PFK2. How?

Not directly, but indirectly.
*in high insulin, PFK2 is NOT phosphorylated
*de-phosph PFK2 is a kinase and it will make F26BP which is the most potent activator of PFK1, which in turn is the important enzyme for formation of F16BP, the committed reaction of glycolysis
*thus, when PFK2 is phosphorylated, glycolysis is inhibitied (PFK2 is a phosphatase when phosphorylated) and gluconeogenesis is promoted
*opposite is true for high insulin state

11

Why is glyceraldehyde-3-phosphate dehydrogenase an important enzyme to recognize?

*not for high regulation, but for the step it is involved in
*(2) glyceraldehyde-3-P + 2 NAD+ --> (2) 1,3-bisphosphoglycerate + 2 NADH
*you now have the first energy payoff of glycolysis!
*this is the first oxidation reaction in glycolysis
*NAD+ must be regenerated to continue (thus lactate production in mitochondria-less cells)

12

What is the first substrate-level phosphorylation reaction in glycolysis?

(2) 1,3-bisphosphoglycerate + 2 ADP --> (2) 3-phosphoglycerate + ATP
*at this point the net ATP is 0 because there were investment reactions before this
*2 ATP produced at this step
*catalyzed by phosphoglycerate kinase

13

When does glycolysis produce net positive ATP?

(2) phosphoenol pyruvate + 2 ADP ---> (2) pyruvate + 2 ATP
*last part of glycolysis cycle
*catalyzed by IMPORTANT enzyme = pyruvate kinase

14

Why is pyruvate kinase an important enzyme to recognize?

*involved in the first step in glycolysis that provides net + ATP
*this is actually the last step of glycolysis, that produces pyruvate
*super regulated b/c either glycolysis or gluconeogenesis
*also, pyruvate is super important hub for other reactions, so pay attention to what makes pyruvate or breaks it
*2nd reaction of substrate-level phosphorylation
*inhibited by phosophorylation (PKA)
*inhibited by alanine
*inhibited by ATP
*stimulated by de-phosphorylation
*stimulated by F16BP
*think insulin and glucagon messing with PKA for this one

15

When you see enzyme-linked hemolytic anemia you think what two deficiencies

*most common is G6PD
*second most common is pyruvate kinase deficiency
*this means that RBCs never get ATP net +, and since they don't have mitochondria, it's hard for them to get energy

16

Lactate dehydrogenase does what?

*it works BOTH directions for conversion of pyruvate to lactate
*uses CoA and CO2
*makes NAD toward lactate
*uses NAD to make NADH toward pyruvate

17

what is happening in the fed vs. fast state in terms of pyruvate handling?

Fed state - pyruvate is used to make other stuff, either fatty acid synthesis or amino acids
Fasting state - pyruvate is made into oxaloacetate and shoved down the TCA cycle

18

Why is the reaction catalyzed by the pyruvate dehydrogenase complex so very important for TCA cycle?

*this is the first step of feeding pyruvate into the TCA cycle
*it makes acetyl-CoA
*it takes place immediately after pyruvate is moved from the cytoplasm to the mitochondria
*dependent on several co-enzymes
*dependent on several vitamins

19

what vitamins are important for the pyruvate dehydrogenase complex?

Thiamine (vitamin B1) - TTP
Riboflavin (vitamin B2) - FAD
niacin - NAD
pantothenate coenzyme A

20

why is thiamine deficiency an example of important glucose handling?

thiamine, or vitamin B1, is super important for the pyruvate dehydrogenase complex to make acetyl-CoA to start the TCA cycle
*if there is little B1, then the brain has trouble with it's main energy producing pathway (oxidation of glucose with TCA and electron transport)
*thus, B1 deficiency presents as wernicke's encephalopathy b/c neurons are whiny when starved
*also can present with beriberi problems in the heart because the heart muscle also uses TCA cycle a ton

21

How is PDH complex regulated (fed vs. fasting)?

*activated when energy is low
*inhibited when energy is high
*NADH, ATP, Acetyl-CoA, Fatty acids all alosterically inhibit the complex
*fasting IN THE LIVER, pyruvate is shunted to the gluconeogenesis pathway by the work of pyruvate carboxylase, which is stimulated by acetyl-CoA presence
*essentially, IN THE LIVER, in a fasting state pyruvate is moved up the chain, not down
*insulin = dephosp state, induction
*glucacon = phosph state, inhibition
*increased clacium means incrased dephosp state as calcium stimulates phosphatase

22

In one turn of the TCA cycle, what leaves and what enters?

1 acetyl group (acetyl-CoA) - 2 carbons that enter
*2 CO2 will leave the cycle
*oxaloacetate is used, but regenerated, leading to no net change in this carbon skelaton

23

What is important about citrate synthase?

*this enzyme is involved in the combining of the 2C acetyl-CoA with the 4C oxalaloacetate into citrate
*irreversible
*citrate is an important allosteric regulator and hub for formation of fatty acids
*allosterically inhibits PFK1 (which is the rate-limiting step in glycolysis, so this is feedback inhibition at its finest)

24

The production of alpha-ketoglutarate is important why?

*catalyzed by isocitrate dehydrogenase
*first CO2 produced
*first NADH produced
*entry point for amino acids to start gluconeogenesis

25

alpha-ketoglutarate dehydrogenase is important why?

*catalyzes the production of succinyl CoA
*complex of enzymes similar to pyruvate dehydrogenase
*5 coenzymes are required
*2nd time for generation of CO2 and NADH
*entry point for amino acids with odd number of carbons

26

where in the TCA cycle do you see SLP?

SLP = substate level phosphorylation
*from succinyl CoA to succinate
*succinate thiokinase - aka - succinyl coa synthetase
*important reaction b/c GTP is formed by substrate level phosphorylation

27

where are all the TCA cycle enzymes?

In the mitochondrial matrix. There is direct interplay between the electron transport chain and the TCA cycle

28

the oxidation of succinate to fumarate is important. WHY?

*enzyme - succinate dehydrogenase
*production of FADH2 from FAD
*this goes immediately and directly into the electron transport chain
*FAD is enzyme bound and is the electron acceptor in this reaction

29

what two "end" products of the TCA cycle are involved in gluconeogenesis?

Malate and OAA (oxaloacetate)

30

Where does the third NADH get generated in the TCA cycle?

oxidation of malate to oxaloacetate
*enzyme - maltate dehydrogenase
*this reaction is important b/c of ties to gluconeogenesis and b/c of the NADH generation