BCEM 393 - Glycolysis, Fermentation, Regulation & Gluconeogenesis

glycolysis

oxidizing carbon sources to release energy

allows glucose to be converted into 2 pyruvate along with 2 ATP and 2 NADH

glucose + 2 ADP + 2 NAD⁺ + 2 Pi —> 2 pyruvate + 2 ATP + 2 NADH + 2 H⁺ + 2 H2O

oxidation

losing e-

reduction

gaining e-

substrate-level phosphorylation

enables net ATP production

hexokinase

step 1 of glycolysis, ATP donates a phosphoryl group to glucose to form glucose 6-phosphate

this traps the glucose molecule in the cell because glucose transporters cannot transport G-6P

phosphoglucose isomerase

step 2 of glycolysis, convert glucose 6-phosphate to fructose 6-phosphate

aldose —> ketose (carbonyl moves from C1 to C2)

fructose can readily by cleaved into 2 3C fragments

phosphofructokinase

step 3 of glycolysis, fructose 6-phosphate becomes fructose 1,6-bisphosphate

ATP donates a phosphoryl group to F-6P (uses 1 ATP)

now molecule has 2 phosphoryl groups

aldolase

step 4 of glycolysis, fructose 1,6-bisphosphate is cleaved into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate

a 6C unit cleaved into 2 3C units, products are isomers

cleaved at bond B to carbonyl (labile bond)

triose phosphate isomerase

step 5 of glycolysis, dihydroxyacetone phosphate is isomerized to glyceraldehyde 3-phosphate

thus at the end of the preparation stage, 1 glucose yields 2 GAPs

glyceraldehyde 3-phosphate dehydrogenase

step 6 of glycolysis, GAP donates e- to NAD+ in a redox reaction forming NADH then Pi is attached to form 1,3-bisphosphoglycerate

NAD+ is reduced and GAP is oxidized

phosphoglycerate kinase

step 7 of glycolysis, 1,3-bisphosphoglycerate donates a phosphoryl group to ADP to form ATP and 3-phosphoglycerate (substrate level phosphorylation)

phosphoglycerate mutase

step 8 of glycolysis, 3-phosphoglycerate becomes 2-phosphoglycerate

phosphoryl group moves from C3 to C2

*mutase looks like an isomerase but there is an enzymatic difference

enolase

step 9 of glycolysis, 2-phosphoglycerate becomes phosphoenolpyruvate

dehydration forms a double bond

pyruvate kinase

step 10 of glycolysis, phosphoenolpyruvate becomes pyruvate

PEP donates a phosphoryl group to ADP to form ATP (2nd substrate level phosphorylation)

glycolysis under cell conditions

negative DG, reaction quotient Q allows for spontaneous reactions

possible fates of pyruvate

1. ethanol fermentation

2. lactic acid fermentation

3. tricarboxylic acid (TCA) cycle

ethanol fermentation

pyruvate —> acetaldehyde —> ethanol

anaerobic (no O2), only in yeast and some bacteria

CO2 released and NAD+ regenerated

lactic acid fermentation

pyruvate —> lactate

anaerobic (no O2), in humans

regenerates NAD+

tricarboxylic acid (TCA) cycle

pyruvate —> acetyl CoA

aerobic (O2)

pyruvate turns into more energy (ATP)

pyruvate decarboxylase

enzyme involved in ethanol fermentation

pyruvate undergoes decarboxylation, releasing CO2 to form acetaldehyde

alcohol dehydrogenase

enzyme involved in ethanol fermentation

acetaldehyde is reduced to ethanol while NADH is oxidized to NAD+

lactate dehydrogenase

enzyme involved in lactic acid fermentation

pyruvate is reduced to lactate while NADH is oxidized to NAD+

NAD+ regeneration

NAD+ consumed in glycolysis needs to be regenerated for glycolysis to continue

fermentation reoxidizes NADH to NAD+ so then NAD+ may continue to fuel glycolysis under anaerobic conditions

needed to maintain redox balance

3 irreversible steps of glycolysis

hexokinase

phosphofructokinase

pyruvate kinase

important for regulation

regulation of glycolysis

mainly regulated at the 3 irreversible steps, regulation by allosteric effectors or covalent modification

occurs differently in different tissues

allosteric regulation

effector molecule binds to allosteric site (not the active site) on the enzyme that can either activate or inhibit the enzyme’s activity

more temporary, diffusible

covalent modification

attachment or removal of chemical group from an enzyme that can either activate or inhibit the enzyme’s activity, eg. phosphorylation

on or off switch

phosphofructokinase (glycolysis regulation in skeletal muscles)

allosterically inhibited by ATP

activated by AMP - during low ATP, AMP is produced

AMP competes with ATP for the binding site, reversing the inhibitory actions of ATP

hexokinase (glycolysis regulation in skeletal muscles)

allosterically inhibited by its own product (glucose 6-phosphate) - negative feedback

when PFK is inhibited, fructose g-phosphate and glucose 6-phosphate accumulates

pyruvate kinase (glycolysis regulation in skeletal muscles)

allosterically activated by fructose 1,6-bisphosphate - feedforward stimulation

allosterically inhibited by ATP

regulation of glycolysis in the liver

glucose can be stored as glycogen in the liver

liver regulates blood glucose levels

high glucose —> stores glucose as glycogen

low glucose —> releases glucose from glycogen stores

phosphofructokinase (glycolysis regulation in liver)

liver does not have urgent ATP needs, regulation by other metabolic signals

citrate as allosteric inhibitor

fructose 2,6-bisphosphate as allosteric activator - feedforward stimulation - abundant fructose 6-phosphate is converted to fructose 2,6-bisphosphate

F-2,6-BP increases PFK’s affinity for F-6P and decreases the inhibitory effects of ATP

F-2,6-BP levels also modulated by enzyme PFK2

hexokinase (glycolysis regulation in liver)

glucokinase (hexokinase IV) isozyme in liver, not inhibited by glucose 6-phosphate

Km for glucose is 50x higher than hexokinase (low affinity), does not catalyze when glucose is limited to ensure that the brain and muscles has first dibs on glucose supply

isozymes

enzymes that catalyze the same reaction but have different primary structures, leading to different regulation and kinetic properties

pyruvate kinase (glycolysis regulation in liver)

inhibited by reversible phosphorylation with ATP

allosterically inhibited by ATP and alanine (derived from pyrvuate) and activated by fructose 1,6-bisphosphate

GLUT2

glucose transporter in liver and pancreatic B cells

pancreas - plays a role in the regulation of insulin

liver - removes excess glucose from the blood

gluconeogenesis

2 pyruvate —> glucose (reverse of glycolysis), mainly occurs in the liver

can also be done with non-carbohydrate molecules (eg. lactate, specific amino acids, glycerol)

mostly in cytoplasm

gluconeogenesis equation

2 pyruvate + 4 ATP + 2 GTP + H+ + 6 H2O —> glucose + 4 ADP + 2 GDP + 6 Pi

pyruvate carboxylase

1 of two enzymes responsible for reversing the pyruvate kinase reaction from glycolysis

carboxylation of pyruvate (3C) to produce oxaloacetate (4C), driven by ATP hydrolysis

occurs inside the mitochondrion

oxaloacetate

produced in gluconeogenesis from pyruvate catalyzed by pyruvate carboxylase

cannot be transported into the cytoplasm from the mitochondrion, must be reduced to malate first

phosphoenolpyruvate carboxykinase

1 of two enzymes responsible for reversing the pyruvate kinase reaction from glycolysis

generates phosphoenolypyruvate from oxaloacetate, removes a CO2, GTP donates phosphoryl group

fructose 1,6-bisphosphatase

removes a phosphoryl group from fructose 1,6-bisphosphate with water to produce fructose 6-phosphate

reverses the phosphofructokinase reaction in glycolysis

glucose 6-phosphatase

reverses the hexokinase reaction from glycolysis, removes a phosphoryl group from glucose 6-phosphate with water

but most tissues stop at glucose 6-phosphate because they want to retain glucose while the liver controls blood glucose levels

NADH in gluconeogenesis

equation does not take into account cytosolic _____ produced during oxaloacetate transport from mitochondrion into cytoplasm

Cori cycle

for regenerating glucose, lactate from anoxic glycolysis in muscles can cycle back in and be released back into the bloodstream as glucose

using stores of glycogen and fatty acids to drive glucose production

regulation of PFK and F-1,6-bisphosphatase

bifunctional regulatory enzyme regulates intercnversion between F6P and F16BP by controlling levels of F26BP

enzyme has two domains - PFK2 and FBPase2

when PFK2 is phosphorylated

PFK2 is inactivated and FBPase2 is activated

when PFK2 is not phosphorylated

PFK2 is activated and FBPase2 is inactivated

when glucose is high

PFK2 is dephosphorylated and becomes active, PFK2 phosphorylates F6P to produce fructose-2,6-bisphosphate, F26BP allosterically activates PFK

stimulates glycolysis and inhibits gluconeogenesis

when glucose is low

PFK2 is phosphorylated and becomes inactive, while FBPase2 is activated, FBPase2 dephosphorylates fructose 2,6-bisphosphate back to fructose 6-phosphate

PFK is not activated by fructose-2,6-bisphosphate

inhibits glycolysis and stimulates gluconeogenesis

pyruvate kinase regulation in the liver

allosterically inhibited by ATP and alanine

activated by fructose 1,6-bisphosphate

inhibited by reversible phosphorylation with ATP when blood glucose is low (regulated reciprocally to PFK2)

diabetes type 2

due to glucose insensitivity

insulin insensitivity downregulates the signaling pathway, yielding high levels of PEPCK and higher gluconeogenesis

metabolic flux

amount of metabolites passing through a metabolic reaction/pathway over time

futile cycle

flux through two opposite metabolic pathways are equal, resulting in no net useful output

will result in the loss of energy

prevented by tight regulation, ensuring only one pathway is active at a time