Biochem I Lecture 26-28

how many ATPs does the metabolic energy of glucose produce?

4

the net yield of ATP for glycolysis is

2 ATP

what are the two high-energy phosphate intermediates in the second phase of glycolysis?

1,3 BPG and PEP

what occurs in rxn 6 of glycolysis?

G3P is converted into 1,3 BPG and NADH

- the only oxidation rxn

- uses inorganic phosphate to provide the phosphoryl group

- involves covalent catalysis and NAD+ coenzyme

- enzyme rxn is the site of arsenate- anion analogous to phosphate

what is arsenate?

substrate for G3P-DH rxn

- forms 1-A-3-P which breaks down into 3-PG

- bypasses phosphoglycerate kinase rxn

- no net ATP

what happens when a sulfhydryl and G3P form a thiohemiacetal?

loses hydride to NAD+ to become a thioester

what occurs in rxn 7 of glycolysis?

1,3-BPG to 3-PG

- ATP synthesis from a high energy phosphate

- phosphoryl group from 1,3-BPG is transferred to ADP to form ATP via PGK

- substrate level phosphorylation

- pays off the ATP debt created by priming rxns in the first phase

what is 2,3-BPG?

- an important regulator of hemoglobin

- side step

- formed from 1,3-BPG by BPG mutase

- 3-PG then formed by 2,3-BPG phosphatase

what is important about the mutate that forms 2,3-BPG from 1,3-BPG?

it requires 3-PG

- intermolecular transfer from C-1 of 1,3-BPG to C-2 of 3-PG

what occurs in rxn 8 of glycolysis?

phosphoglycerate mutase catalyzes phosphoryl group transfer from C3 to C2

- PG mutase- catalyzes migration of a functional group within a substrate (mimics enol)

- repositioning the phosphate to set up the making of PEP

- intramolecular

- small amount of 2,3-BPG is used to phosphorylate the histidine residue, thus forming a histidine intermediate

what occurs in rxn 9 of glycolysis?

conversion of 2-PG into 2 PEP via enolase

- creates a high-energy phosphate in preparation for ATP synthesis in rxn 10

- 2-PG and PEP have similar energy content, but PEP is formed to release MORE energy during hydrolysis

what occurs in rxn 10 of glycolysis?

conversion of PEP to pyruvate to make ATP via pyruvate kinase

- two ATP = "payoff" of glycolysis

- large, negative ∆G value, so it's spontaneous and can't be reversed

PK is allosterically activated by

AMP and F-1,6-bisP (committed step 3)

PK is allosterically inhibited by

ATP and acetyl-CoA (and alanine)

what are the two steps of converting PEP into pyruvate?

1. phosphoryl transfer

2. enol-keto tautomerization (spontaneous)

how is Liver PK phosphorylated?

by protein kinase

- activated by glucagon which signals glucose status

- when phosphorylated, it has a higher Km value for PEP, so it's more strongly inhibited by ATP and alanine

what are the 2 possible fates of NADH?

- aerobic: NADH is oxidized in the electron transport pathway, making ATP in oxidative phosphorylation

- anaerobic: NADH is oxidized by lactate dehydrogenase, providing additional NAD+ for more glycolysis (recycling)

what happens in oxygen-depleted muscle?

NAD+ is regenerated in the lactate dehydrogenase rxn

- too much lactate can cause cramps

what occurs in hibernating turtles?

they become anoxic

- convert glucose into lactate

- they have minerals in their shells which buffer lactate

what is another process in which lactate is produced?

lactic-acid bacteria

- produces acid flavor in yogurt and sauerkraut

what are the 2 metabolic fates of pyruvate?

- aerobic: TCA cycle

- anaerobic: lactate (or ethanol in yeast)

what is fermentation?

production of ATP by rxn pathways in which organic molecules function as donors AND acceptors of electrons

does the conversion of glucose to lactate involve any net oxidation or reduction?

no

- although, enough energy is liberated to sustain growth

what were butanol and acetone made by?

clostridium acetobutylicum

- marked the beginning of modern biotechnology

which 3 rxns are sites of regulation?

rxn 1: hexokinase

rxn 3: PFK

rxn 10: pyruvate kinase

- they all have very large negative ∆G values

- they can turn glycolysis on and off as they are key points in which glycolysis is regulated

how was acetone made?

fermenation and used in the manufacture of cordite during WWI

Entner-Doudoroff pathway

- found only in some bacteria

- tequila fermentation

- makes more ethanol and tolerates higher ethanol concentrations than yeast, making it of industrial interest

what attracts interest as a possible fuel source?

butanol

what is the Leloir pathway?

converts galactose to glucose

what are some other sugars other than glucose that can enter the glycolytic pathway?

mannose, galactose, and fructose

how is glycerol produced?

in the decomposition of triacylglycerols (TAGs)

how can glycerol be converted to glycerol-3-P?

glycerol kinase

- it is then oxidized into dihydroxyacetone phosphate (DHAP) via glycerol phosphate dehydrogenase

where and what is lactose made by?

it is made by lactose synthase and it occurs in the mammary gland during late pregnancy and lactation

what is lactose synthase?

a dimeric complex of galactose transferase and α-lactalbumin

what provides newborns with essential galactose?

lactose breakdown in intestine by lactase

why does lactose intolerance occur?

low level of lactase

what is gluconeogenesis?

synthesis of glucose from small molecule precursors

- good example of how competing catabolic and anabolic pathways are regulated, so each one is only active when required

are anabolic pathways the reverse of the catabolic pathway?

NO, but some rxns are shared (not 1, 3, and 10)

glucose needs in the body

- humans: 160 grams of glucose/day

- body fluids: 20 grams of free glucose, 200 grams as glycogen

- glycogen stores at least partially depleted in strenuous exercise

- body carries a day supply of glucose. Obtained via diet if insufficient, then synthesis of new glucose from non-carbohydrate precursors are necessary

- body must make its own glucose to restore amount stored in glycogen to sustain normal activity

does fat catabolism generate glucose?

no

how are pyruvate and lactase produced and recycled?

produced during exercise, recycled to glucose by gluconeogenesis

what all can be converted to glucose via gluconeogenesis?

pyruvate, lactate, most amino acids, glycerol, and citric acid intermediates

major site for glucose consumption

brain and muscle

where is most of gluconeogenesis accounted for?

liver and kidney

why can't gluconeogenesis be the simple reverse of glycolysis?

- glycolysis is exergonic

- different enzymes are required

- reciprocal regulation of glycolysis and gluconeogenesis

in rxn 10 of gluconeogenesis, what enzymes replace pyruvate kinase?

pyruvate carboxylase and PEP carboxykinase

in rxn 3 of gluconeogenesis, what enzyme replaces PFK?

fructose-1,6-bisphosphate

in rxn 1 of gluconeogenesis, what enzyme replaces hexokinase?

glucose-6-phosphatase

what is reciprocal regulation of glycolysis and gluconeogenesis?

when glycolysis is active, gluconeogenesis is turned off and vice versa

what can NOT be used as a substrate in gluconeogenesis?

fatty acids

- Acetyl-CoA (via TCA cycle) cannot provide for net synthesis of sugars

are ATP and GTP the same?

yes

what does pyruvate carboxylase do?

converts pyruvate to oxaloacetate

- occurs in mitochondria since oxaloacetate cannot be transported across the mitochondrial membrane, so it must be reduced to malate

- requires ATP

what does PEP carboxykinase do?

Converts oxaloacetate to PEP

- driven forward via decarboxylation or hydrolysis of GTP

what does fructose-1,6-bisphosphatase do?

removes a phosphate from fructose 1,6-bisphosphate to produce fructose 6-phosphate

What does glucose-6-phosphatase do?

converts glucose-6-phosphate to glucose

what is required for pyruvate to be converted into oxaloacetate?

ATP and bicarbonate

- bicarbonate used as the carbon source, thus requiring biotin as a coenzyme

what is biotin?

- carboxyl group carrier

active-site lysine

what's an allosteric activator of pyruvate carboxylase?

acetyl-CoA, created via TCA cycle

what's involved in the regulation of pyruvate carboxylase?

when ATP or acetyl-CoA are high, pyruvate enters gluconeogenesis

when bicarbonate is activated, what happens?

attack by pyruvate carbanion

what's the sequence of pyruvate carboxylase?

1. attack of HCO3 on ATP

2. formation of carbonylphosphate

3. formation of N-carbonyl biotin

5. release of Pi

6. -3 carbanion of pyruvate attack

what does malate do?

transported to the cytosol and oxidized back to oxaloacetate before gluconeogenesis can continue

what does compartmentalization help with?

preventing simultaneous glycolysis and gluconeogenesis

what does transport of oxaloacetate to cytoplasm require?

conversion of malate by NADH-linked malate hydrogenase

what happens if acetyl-CoA is low?

carbon is channeled towards the citric acid cycle, inhibiting gluconeogenesis

what happens if acetyl-CoA is high?

carbon is channel towards gluconeogensis, and citric acid cycle is inhibited

- same is true if there are high levels of ATP

what do high levels of acetyl CoA and ATP indicate?

high energy status, thus turning on gluconeogenesis

what is GTP hydrolysis equivalent to?

consumption of ATP, since it is required to phosphorylate GDP to give GTP

what is the overall ∆G value for the formation of PEP from pyruvate?

-22.6 kJ/mol

what is favored in fructose-1,6-bisphosphatase?

phosphate ester hydrolysis

what is fructose-1,6-bisphosphatase activated and inhibited by?

- activated by citrate

- inhibited by fructose-2,6-bisphosphate and AMP (activates PFK)

- reciprocal regulation of glycolysis and gluconeogenesis occurs at the enzymatic steps NOT common in the two pathways

is Hussein's haircut fire?

no

PFK-2 and F-2,6-BPase are two activities of a ________ , or _________ enzyme.

bifunctional, tandem

what does F-6-P do?

allosterically activates PFK-2

what does PFK-2 do?

makes F26BP from F6P

- this acts as an allosteric activator for PFK-1 and allosteric inhibitor for F-1,6-BPase

- pushes glycolytic pathway forward

what is responsible for the phosphorylation of PFK-2, and what does this do?

PFK-2 is phosphorylated by cAMP dependent protein kinase, and this converts it into fructose-2,6-bisphosphatase

what does glucagon do?

detects low glucose

- activates protein kinase A, which activates FBPase-2 making gluconeogenesis more active

- F26BP to F6P

what does insulin do?

detects high glucose

- activates protein phosphatase-1 which activates PFK-2 making glycolysis more active

- F6P to F26BP

G-6-P is

exergonic via phosphate ester hydrolysis

- nucleophilic attack by histidine nitrogen

- formation of phosphohistidine intermediate

where is G6P converted into glucose and by what?

glucose-6-phosphates regulates this conversion in the endoplasmic reticulum

as 2 pyruvate are converted to glucose, what is hydrolyzed?

six nucleoside triphosphates (4 ATPs and 2 GTPs)

- makes gluconeogenesis exergonic and thermodynamically feasible

how does the liver help us during exercise?

Cori Cycle

- buildup of lactate and NADH due to oxygen shortage and need for more glycolysis

- NADH is deoxidized during reduction of pyruvate to lactate

- lactate returned to liver where it can be deoxidized to pyruvate by liver LDH (isozyme)

- liver provides glucose to muscle for exercise and reprocesses lactate into new glucose

what would happen without reciprocal control of glycolysis and gluconeogenesis?

futile cycle- massive ATP consumption

what happens when energy status is low?

glucose is degraded to provide ATP

what happens when energy status is high?

pyruvate is used for synthesis of glucose then glycogen

when glycolysis is turned on,

gluconeogenesis is turned off

when gluconeogenesis is turned on,

glycolysis is turned off

which steps are regulated in both gluconeogenesis and glycolysis?

1,3, and 10

glucose-6-phosphatase activity increases linearly with

substrate concentration

- substrate level control

acetyl-CoA inhibits ______ and stimulates ______

glycolysis, gluconeogenesis

- high acetyl-CoA = high energy status (TCA cycle)

high AMP signals

low energy status, and stimulates glycolysis and

inhibits gluconeogenesis

reciprocal regulation by fructose-2,6-bisphosphate activates

PFK-1, stimulating glycolysis