Fundamentals of Biochemistry

Part III => METABOLISM and ENERGY
3.2 GLUCOSE CATABOLISM
3.2a Glycolysis Pathway
3.2b Glycolysis Regulation
3.2c Fermentation
Section 3.2a:
Glycolysis
Synopsis 3.2a
- Dietary starch (eg bread, rice and potatoes) is hydrolyzed into glucose by the
combined action of enzymes such as amylase (saliva) and maltase (small intestine)
- Glycolysis involves the breakdown/oxidation of glucose into pyruvate using a wide
array of enzymes—and the free energy released in the process is used to
synthesize ATP from ADP
- In terms of chemical reactions, the glycolytic enzymes catalyze phosphorylation
(transferase), isomerization (isomerase), bond cleavage (lyase), dehydrogenation
(oxidoreductase), and hydrolysis (hydrolase)
- Of the six major classes/families of enzymes (§2.5), all but ligase are involved in
mediating glycolysis!
- The 10-reaction sequence of glycolysis is divided into two stages:
Stage I  Energy investment/expenditure
Stage II  Energy recovery/payoff
- Overall glycolytic reaction:
Glucose + 2NAD+ + 2ADP + 2Pi <=> 2Pyruvate + 2NADH + 2ATP + 2H2O + 2H+
Glycolysis
Overview
Glycolysis can be divided
into two main stages
G / kJ.mol-1
-34
0
-19
0
Stage I (Steps 1-5)
0
0
Stage II (Steps 6-10)
0
0
0
Glycolysis is accompanied
by a net G of -76 kJ per
mole of glucose converted
to two moles of pyruvate
-23
-76
Glycolysis Stage I (Investment): Glucose  GAP
1
2
In Stage I (Steps 1-5):
- 1 molecule of glucose is converted
to 2 molecules of glyceraldehyde-3phosphate (GAP)
3
- 2 molecules of ATP are utilized
(energy investment)
4
5
(1) Glucose  Glucose-6-Phosphate
G = -34 kJ/mol
- Transfer of the terminal phosphoryl group of ATP to glucose to generate G6P
- Thermodynamically favorable—powered by the free energy released due to ATP
hydrolysis!
- Catalyzed by hexokinase (HK)—a non-specific enzyme that not only catalyzes the
phosphorylation of glucose but also other hexoses such as mannose and fructose
- As is true for kinases in general, hexokinase
requires Mg2+ divalent ions for catalytic activity—
the Mg2+ ion is believed to shield the negative
charges on - and -phosphate oxygen atoms
within ATP, so as to render its -phosphate
atom more accessible to nucleophilic attack by
the –CH2OH group of glucose
(2) Glucose-6-Phosphate  Fructose-6-Phosphate
G
- Isomerization of 6-membered
G6P to 5-membered F6P
- Thermodynamically neutral—
operates near equilibrium!
- Catalyzed by phosphoglucose
isomerase (PGI)
- Requires ring opening of G6P
followed by isomerization and
subsequent ring closure to
generate F6P
0 kJ/mol
(3) Fructose-6-Phosphate  Fructose-1,6-Bisphosphate
G = -19 kJ/mol
- Phosphorylation of F6P to FBP—a rate-determining step of glycolysis
- Thermodynamically favorable—thanks to the free energy provided by ATP hydrolysis!
- Catalyzed by phosphofructokinase (PFK) in a manner akin to phosphorylation of
glucose to G6P by hexokinase (Step 1)—requires Mg 2+ ion as a cofactor!
- PFK serves as a key regulatory player in glycolysis—its catalytic activity is allosterically
enhanced by AMP/ADP and inhibited by ATP (vide infra)!
- Note that bisphosphate refers to the attachment of two phosphate groups to separate
moieties in lieu of directly to each other—which would be diphosphate as in ADP
(4) Fructose-1,6-Bisphosphate  GAP + DHAP
+
G
0 kJ/mol
- Cleavage of a single 6-C compound (FBP) into two 3-C compounds (GAP and DHAP)
- Thermodynamically neutral—operates near equilibrium!
- Catalyzed by aldolase (ALD) into two interconvertible 3-C compounds—aldolase
belongs to the lyase family of enzymes that catalyze breaking/elimination of bonds by
means other than hydrolysis!
- Note that the atom nomenclature changes upon cleavage of FBP—atoms 4/5/6 in FBP
become atoms 1/2/3 in GAP, while atoms 1/2/3 in FBP become atoms 3/2/1 in DHAP
(5) Dihydroxyacetone Phosphate  Glyceraldehyde-3-Phosphate
triose
phosphate
isomerase
[DHAP]
G
0 kJ/mol
[GAP]
- Interconversion of DHAP to GAP via an enediol intermediate
- Thermodynamically neutral—operates near equilibrium!
- Catalyzed by triose phosphate isomerase (TIM)—a “perfect” enzyme in that it
operates near the diffusion-controlled limit with kcat/KM 109 M -1s-1 (§2.6)
- DHAP and GAP are ketose-aldose isomers
- Only GAP continues along the glycolytic pathway—as GAP is siphoned off, more DHAP
is converted to GAP due to “equilibrium shift”!
Glycolysis Stage II (Recovery): GAP  Pyruvate
6
In Stage II (Steps 6-10):
- 2 molecules of GAP are converted to 2
molecules of pyruvate with concomitant
generation of high-energy compounds (ATP
and NADH) that can be used as energy
currencies right away
7
8
- 4 molecules of ATP via substrate-level
phosphorylation
- 2 molecules of NADH via reduction of NAD+
9
10
(6) Glyceraldehyde-3-phosphate  1,3-Bisphosphoglycerate
HPO42-
G
0 kJ/mol
- Oxidation and phosphorylation of GAP to “high-energy” 1,3-BPG with concomitant
release of NADH— cf the “high-energy” nature of mixed anhydride bond in 1,3BPG vs the terminal phosphoanhydride bond in ATP!
- Hydrogen phosphate (HPO42-) used as a phosphate donor
- NAD+ used as an oxidizing agent to oxidize GAP
- Thermodynamically neutral—operates near equilibrium!
- Catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(7) 1,3-Bisphosphoglycerate  3-Phosphoglycerate
G
0 kJ/mol
- Dephosphorylation of 1,3-BPG to 3PG
- “High-energy” 1,3-BPG used as a phosphate donor to drive the synthesis of ATP
from ADP via substrate-level phosphorylation (first ATP generation)
- Thermodynamically neutral—in spite of the free energy provided by the hydrolysis
of “high-energy” 1,3-BPG, the reaction operates near equilibrium!
- Catalyzed by phosphoglycerate kinase (PGK)—requires Mg 2+ ion as a cofactor!
(8) 3-Phosphoglycerate  2-Phosphoglycerate
G
0 kJ/mol
- Isomerization of 3PG to 2PG in an intramolecular transfer of phosphate group
- Thermodynamically neutral—operates near equilibrium!
- Catalyzed by phosphoglycerate mutase (PGM)
- What do you call an isomerase that catalyzes the intramolecular transfer of a
functional group from one position to another? Mutase!
(9) 2-Phosphoglycerate  Phosphoenolpyruvate
G
0 kJ/mol
- Dehydration of 2-phosphoglycerate to generate “high-energy” phosphoenolpyruvate
(or 2-phosphoenolpyruvate)—cf the “high-energy” nature of phosphoester bond in
PEP (the highest-energy phosphate bond in nature) vs the terminal
phosphoanhydride bond in ATP!
- Thermodynamically neutral—operates near equilibrium!
- Catalyzed by enolase—an enzyme that belongs to the lyase family of enzymes that
catalyze breaking/elimination of bonds by means other than hydrolysis!
(10) Phosphoenolpyruvate  Pyruvate
G = -23 kJ/mol
- Dephosphorylation of phosphoenolpyruvate (or 2-phosphoenolpyruvate) to
pyruvate– or systematically, -ketopropionate
- “High-energy” PEP used as a phosphate donor to drive the synthesis of ATP
from ADP via substrate-level phosphorylation (second ATP generation)
- Thermodynamically favorable—thanks to the free energy provided by the
hydrolysis of high-energy PEP!
- Catalyzed by pyruvate kinase (PK)—requires Mg2+ (or Mn2+) ion as a cofactor!
Exercise 3.2a
- What happens during the two phases of glycolysis?
- How many ATP molecules are invested and how many are recovered
from each molecule of glucose that follows the glycolytic pathway?
- Write the reactions of glycolysis, showing the structural formulas of the
intermediates and the names of the enzymes that catalyze the reactions.
Distinguish between thermodynamically neutral and favorable steps.
- Summarize the types of catalytic mechanisms involved. Do any glycolytic
enzymes require cofactors?
- What “high-energy” compounds are synthesized during glycolysis?
Section 3.2b:
Glycolysis Regulation
Synopsis 3.2b
- Enzymes that function with large negative G are
candidates for flux-control points
- Phosphofructokinase (PFK), the major regulatory point
for glycolysis in muscle, is allosterically inhibited by
ATP and activated by AMP/ADP
- Substrate cycling allows the rate of glycolysis to
respond rapidly to changing needs
Thermodynamics of Glycolysis (in erythrocytes)
Step Enzyme Name
Enzyme Family
G / kJ.mol-1
G / kJ.mol-1
1
Hexokinase (HK)
Transferase
-17
-34
2
Phosphoglucose isomerase (PGI)
Isomerase
+2
0
3
Phosphofrucktokinase (PFK)
Transferase
-14
-19
4
Aldolase (ALD)
Lyase
+24
0
5
Triose Phosphate Isomerase (TIM)
Isomerase
+8
0
6
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Oxidoreductase
+6
0
7
Phosphoglycerate kinase (PGK)
Transferase
-19
0
8
Phosphoglycerate mutase (PGM)
Isomerase
+4
0
9
Enolase (ENO)
Lyase
+2
0
10
Pyruvate kinase (PK)
Transferase
-32
-23
Garrett R & Grisham CM (2005). Biochemistry (3rd ed). Belmont, CA: Thomson Brooks/Cole. pp 582–583.
- Recall that G = G + RT lnKeq (§1.1)—where G is the actual free energy change under
non-equilibrium (steady-state) conditions, and G is the standard free energy change @
equilibrium!
- Since living cells operate under steady-state rather than equilibrium setting, the free
energy changes associated with various glycolytic steps are largely concerned with G!
- Of the 10 steps of glycolysis, only three (Steps 1/3/10) operate far from equilibrium ( G
<< 0)—implying that they COULD be largely responsible for flux control!
G Profile for Glycolysis
Only Steps 1, 3 and 10 are associated
with large negative G!
PFK Is the Major Flux-Controlling Enzyme
- Actual free energy changes ( G) associated with various glycolytic
steps suggest that the major candidates for flux control are:
- Hexokinase (HK)
=> Step 1
- Phosphofructokinase (PFK) => Step 3
- Pyruvate kinase (PK)
=> Step 10
- Of these three enzymes, only PFK plays a central role
in controlling the rate of flow of metabolites (or flux)
through glycolysis—why?!
- HK is not absolutely critical for glycolysis (much less serve
as a regulatory point!)—since the breakdown of glycogen
into glucose-6-phosphate (G6P) via glycogenolysis, as is often
the case in skeletal muscle, does not require HK (§3.3)
- On the other hand, PK catalyzes the final step of glycolysis—thus its ability to
control flux through glycolysis becomes somewhat moot
- Simply put, the conversion of frucose-6-phosphate (F6P) to fructose-1,6-bisphosphate
(FBP) in Step 3 by PFK is the major rate-determining or rate-limiting (or the slowest)
step of glycolysis
- How is PFK regulated? Allosterically, of course!
Allosteric Regulation of PFK
In respiring muscle:
PFK
Activity
[ATP]/[ADP] ~ 10:1
F6P / mM
- PFK is allsoterically inhibited by ATP—a built-in control to ensure that when ATP is in
excess (there is little demand for energy production), glycolysis is shut off!
- On the other hand, AMP/ADP serve as allosteric activators of PFK—rising cellular
concentrations of AMP/ADP are indicative of energy shortage and thus serve as signals
for the production of ATP
- Acting in concert, AMP/ADP and ATP allsoterically modulate the enzymatic activity of
PFK—thereby enabling PFK to play a key role in the control of flux through glycolysis
Substrate Cycling Fine-Tunes Flux control
3
In resting muscle—
both PFKase and FBPase
are active => low flux
3
In active muscle—
PFKase is active but FBPase
inhibited => high flux
- In addition to its allosteric modulation, the Step 3 of glycolysis is further attuned in
many mammalian tissues via substrate cycling:
- F6P is cycled forth to FBP by phosphofrucktokinase (PFKase)
- FBP is cycled back to F6P by fructose-1,6-bisphosphatase (FBPase)—a hydrolase!
- Substrate cycling can thus potentially decrease the flux through glycolysis by essentially
placing a metabolic intermediate (F6P) into a “holding pattern” until the demand rises!
Exercise 3.2b
- Which glycolytic enzymes are potential control points?
- Describe the mechanisms that control phosphofructokinase activity
- What is the metabolic advantage of a substrate cycle?
Section 3.2c:
Fermentation
Synopsis 3.2c
- Under aerobic conditions, the glycolytic end-product
pyruvate is completely oxidized to CO2 and H2O via the citric
acid cycle (§3.5)
- Under anaerobic conditions, the glycolytic end-product
pyruvate is converted (or reduced) to either lactate or
ethanol in a metabolic process referred to as “fermentation”
- Fermentation can be classified into two major groups:
(1) Homolactic fermentation (in muscle)
(2) Alcoholic fermentation (in yeast)
- In each case, NADH is reoxidized to NAD+ to ensure glycolytic
continuity—thus fermentation plays a key role in the
regeneration of energy under anaerobic conditions!
Metabolic Fate of Pyruvate
O2
Homolactic Fermentation (in muscle)
- During strenuous activity when oxygen is in short supply,
ATP is largely synthesized via anaerobic glycolysis in muscles
- The pyruvate end-product of glycolysis is reduced to lactate
in order to regenerate NAD+ (from NADH)—which is required
for the continuity of anaerobic glycolysis—in a process known
as “homolactic fermentation”
- Homolactic fermentation is catalyzed by lactate dehydrogenase (LDH)
- Under resting conditions when oxygen is no longer limiting, lactate is converted back to pyruvate via
“equilibrium shift” to allow its complete oxidation via the citric acid cycle
- When transported to the liver via the bloodstream, lactate can also be used to synthesize glucose via
gluconeogenesis—generation of glucose from noncarbohydrate sources (including amino acids)
- In anaerobic glycolysis, glycolysis and homolactic fermentation are essentially coupled in that glucose is
oxidized to lactate in a seamless fashion—a virtue of erythrocytes (§3.1)
Alcoholic Fermentation (in yeast)
- Under anaerobic conditions, the major source of ATP in yeast
is also obtained via anaerobic glycolysis
- The pyruvate end-product of glycolysis is ultimately converted
via a two-step process called “alcoholic fermentation” to ethanol:
(1) Pyruvate decarboxylase (PDC) decarboxylates pyruvate to acetaldehyde
using thiamine pyrophosphate (TPP) as a cofactor with concomitant release
of CO2—which serves as the leavening agent in bread
(2) Alcohol dehydrogenase (ADH) subsequently reduces acetaldehyde to
ethanol (the active ingredient in wine and beer) with concomitant
regeneration of NAD+ (from NADH)—which is required for the continuity
of anaerobic glycolysis
- Due to the release of CO2 as a by-product, alcoholic fermentation
is commercially exploited in the production of alcoholic beverages
as well as in the rising of bread (leavened bread)
Exercise 3.2c
- Describe the three possible fates of pyruvate
- Compare homolactic and alcoholic fermentation in terms of the
products and the cofactors required