25 mar 2008 glycolysis andy howard introductory biochemistry 25 march 2008
TRANSCRIPT
25 Mar 2008
Glycolysis
Andy HowardIntroductory Biochemistry
25 March 2008
25 Mar 2008 Glycolysis p. 2 of 56
What we’ll discuss
Glycolysis Overview Steps through
TIM Steps to
pyruvate Fate of pyruvate
Glycolysis (continued) Free energy Regulation Other sugars Entner-Doudoroff
Pathway*
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Glycolysis Now we’re ready for the specifics of
metabolism Why glycolysis first?
Well-understood (?) early on Illustrates concepts used later Inherently important
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The big picture
Conversion of glucose to pyruvate Catabolic, ten steps, energy-yielding Overall reaction:glucose + 2 ADP + 2 NAD+ + 2Pi
2pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
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Significance
Why is this important? Energy production
(ATP and NADH) Pyruvate as precursor to various
metabolites
Some steps require energy So it isn’t all energy-yielding The net reaction yields energy
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The reactions
See fig. 11.2 and the table in the HTML notes
Wide variety of enzyme sizes Most structures have been
determined by X-ray crystallography
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The pathway through TIM
Fig. courtesy U.Texas
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Pathway to pyruvate
Bottom half of same graphic
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Hexokinase Transfers γ-phosphoryl group of
ATP to oxygen atom at C-6 of glucose, producing glucose 6-phosphate and ADP.
Coupling between ATP hydrolysis and an energy-requiring reaction is very close: phosphate is transferred directly from ATP to the recipient molecule, in this case glucose.
The reaction catalyzed by hexokinase is energetically favored: Go’ = -22.3 kJ/mol
G-6-P
PDB 2YHX Yeast52kDa monomer
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Hexokinase isozymes various isozymes (functionally related
but structurally slightly distinct) forms of hexokinase in humans
liver form has Km in millimolar range, perhaps a factor of 1000 higher than the Km of hexokinase found in other tissue
Liver form is therefore much less active than the other forms unless the liver glucose concentration is high
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Activity and complexity
Hexokinase is active on sugars besides glucose;activity against mannose is comparable to the activity on glucose
Hexokinase has the highest molecular mass per monomer of any of the glycolytic enzymes; given that it is the first enzyme in an important pathway, it makes sense that it is large and complex.
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Phosphoglucomutase Interconverts phosphorylated
forms of glucose—glucose 1-P and glucose 6-P.
Intermediate is bisphosphorylated
equilibrium between the 1-P and 6-P forms is determined by relative concentrations.
Active on other phosphorylated aldoses in addition to glucose.
This enzyme doesn’t appear on the chart:not part of the linear pathway from glucose to pyruvate.
PDB 1ZOLLactococcus24 kDa monomer
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Glucose 6-phosphate isomerase
interconverts two monophosphorylated sugars—glucose 6-phosphate and fructose 6-phosphate.
Interconversion proceeds through (1,2) ene-diol intermediate
with enzyme present the energy barriers around this ene-diol are lowered enough to speed the interconversion.
Also called phosphohexoseisomerase or phosphoglucose isomerase
F-6-P
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Properties of G6P isomerase
Dimeric enzyme plays roles extracellularly as well as intracellularly: it can function as a nerve growth factor.
Each monomer contains two unequal-sized domains, and the active site is formed by the association of the two subunits.
PDB 1U0Fmouse124 kDa dimer
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Phosphofructokinase-1 catalyzes phosphorylation at the 1
position of fructose 6-phosphate. example of a kinase that acts on an
already-phosphorylated form, creating a bisphosphorylated compound.
ADP sometimes acts as an allosteric activator on this enzyme as well as being a product of the reaction.
We’ll discuss PFK-2 later
F-1,6-bisP
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PFK-1 structures
Of all the enzymes in this pathway it appears to be the one for which the least structural information is available
Best structure determined to date for the allosteric enzyme was Phil Evans's 2.4 Å structure from 1988, and there have not been many other structures done.
PDB 4pfkE.coli140 kDa tetramer
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Lactobacillus PFK
This one isn’t allosteric No MgADP binding
observed (> 20 mM) Yet it’s highly
homologous! Effector binding site is
very different
PDB 1zxxLactobacillus bulgaricus140 kDa tetramer
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Aldolase Catalyzes actual C-C bond cleavage:
fructose 1,6-bisphosphate D-glyceraldehyde-1-phosphate + dihydroxyacetone phosphate
large and important enzyme Some bacterial and yeast forms require a
divalent cation as a cofactor;eukaryotic aldolases do not.
The non-cationic forms proceed through an imine (Schiff-base) intermediate.
+
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Secondary activity
Enzyme is active on fructose 1-phosphate as well as its "standard" substrate, fructose 1,6-bisphosphate; in this context it forms part of catabolic pathway by which fructose itself can be used as an energy and carbon source.
PDB 1zahRabbit muscle80 kDa dimer
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Triosephosphate isomerase
Interconverts two 3-C phosphosugars
possibly the most efficient enzyme known, in terms of the rate acceleration afforded by the enzyme relative to the uncatalyzed reaction.
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TIM Barrels TIM is an enzyme with a
characteristic structure in which alpha helical stretches alternate with beta strands such that the beta strands curve around to form a barrel-like structure with the helices outside.
This structural motif appears in many other enzymes, and has become known as a "TIM barrel."
PDB 1YPISaccharomyces27 kDa monomer
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Glyceraldehyde 3-phosphate dehydrogenase
medium-sized dimeric or tetrameric enzyme
responsible for the conversion of Glyc-3P to 1,3-bisphosphoglycerate.
Somewhat allosteric PDB 1GD1Bacillus stearothermophilus74 kDa dimer
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Phosphoglycerate kinase
catalyzes dephosphorylation of1,3-bisphosphoglycerate to 3-phosphoglycerate with production of ATP from ADP
named for reaction running in opposite direction relative the one shown in chart.
In the direction shown in the table it produces ATP rather than consuming it.
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PGK Structural Notes Has a hinge motion about a
point near the center of the molecule; the open and closed forms of the enzyme involve movements as large as 17Å in the residues farthest from the hinge point.
Enzyme is primarily alpha-helical in conformation.
PDB 16pkTrypanosoma brucei184 kDa tetramer;monomer shown
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Phosphoglycerate mutase
interconverts 3-phosphoglycerate and 2-phosphoglycerate
Mechanism of reaction involves formation of 2,3-bisphosphoglycerate via transient phosphorylation of a histidine residue of the enzyme.
2-phosphoglycerate
PDB 1e59E.coli55 kDa dimer;monomer shown
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PG Mutase: a problem!
2,3BPG can diffuse from phosphoglycerate mutase, however, leaving the enzyme trapped in an unusable state.
Cells make excess 2,3BPG (using the enzyme bisphosphoglycerate mutase) in order to drive 2,3BPG back to phosphoglycerate mutase, so the reaction can go to completion.
2,3-bisphosphoglycerate
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Enolase interconverts 2-
phosphoglycerate & phosphoenolpyruvate
This reaction plays a role in gluconeogenesis as well as glycolysis.
PDB 4enlSaccharomyces97 kDa dimer; monomer shown
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Enolase details
Mg2+ ions are required for activity, at least in some forms of the enzyme.
Vertebrate genes code for two slightly different forms of the monomer of enolase, alpha and beta.
Most of the enolase in fetal tissue is alpha-alpha; mature skeletal muscle contains beta-beta; some alpha-alpha remains in smooth muscle tissue.
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Pyruvate Kinase transfers a phosphate
from phosphoenolpyruvate to ADP, producing pyruvate and ATP
The reaction is essentially irreversible(Go’ ~ -30 kJ mol-1)
Fructose 1,6-bisphosphate, the substrate for the aldolase reaction, is a feed-forwardactivator of the reaction
PDB 1PKMCat muscle236 kDa tetramermonomer shown
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So we’ve gotten to pyruvate This is conventionally seen as the
endpoint of glycolysis It’s worthwhile, though, to see what can
happen to the products Pyruvate (memorize that structure!) is an
important intermediate in several pathways
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What happens to pyruvate? Four paths:
Pyruvate + CoASH acetylCoA + CO2;this leads to Krebs cycle, to fatty acid biosynthesis, and amino acids
Pyruvate + CO2 oxaloacetate;this is an anapleurotic mechanism for Krebs cycle
Pyruvate + NADH + H+ lactate + NAD+
Pyruvate + H+ acetaldehyde + CO2
acetaldehyde + NADH + H+ ethanol + NAD
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Pyruvate to Lactate Lactate dehydrogenase catalyzes
pyruvate + NADH + H+ lactate + NAD+
Occurs in some anaerobic bacteria and in mammals (e.g. in muscles) if oxygen is not plentiful: anaerobic glycolysis
Net glycolysis reaction under these conditions:glucose + 2 Pi
2- + 2 ADP3- 2 lactate- + 2 ATP4- + 2H2O
Can result in drop in blood pH until reverse reaction (in liver) restores pH and regenerates pyruvate
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Lactate dehydrogenase
Typical tetrameric Rossmann-fold NAD-dependent dehydrogenase
Structural homology to other NAD-binding enzymes
PDB 1xivPlasmodium140 kDa tetramer
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Pyruvate to ethanol
Pyruvate decarboxylated to acetaldehyde:pyruvate + H+ acetaldehyde + CO2
Acetaldehyde is reduced to ethanol:acetaldehyde + NADH + H+ ethanol + NAD
Net glycolytic reaction isglucose + 2 Pi
2- + 2 ADP3- + 2H+ 2 ethanol + 2CO2 + 2 ATP4- + 2H2O
Yeast depend on this pathway
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Pyruvate decarboxylase
Catalyzes first reaction in pathway to ethanol
TPP-dependent reaction: see section 7.7, especially fig. 7.15
Related to the pyruvate dehydrogenase complex that we will meet in chapter 13
PDB 1pvdSaccharomyces62 kDa monomer
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Alcohol dehydrogenase
Second reactionin fermentation path
Reaction itself is reversible:ethanol acetaldehyde direction leads to detox in humans
Often unselective: can be used to oxidize other primary alcohols
PDB 2hcySaccharomyces156 kDa tetramer
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Free energy in glycolysis
Cliché:G matters, not Go’!
See fig. 11.11:Several reactions are endergonic as far as Go’ are concerned, but they’re flat or exergonic with G.
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Hamori’s data
E. Hamori (1975) J.Chem.Ed. 52: 370 Individual values in kcal mol-1
Cumulative values in kJ mol-1
Step Reactant Products DGo' DG Cum DGo' Sum DG0 0 0 0 01 Glucose, ATP G6P, ADP -5.1 -9.5 -21.3 -39.72 G6P F6P 0.49 -0.06 -19.3 -40.03 F6P+ATP FDP + ADP -4.3 -6.2 -37.3 -65.94 FDP 2 Glyc-3P 7.4 -0.17 -6.3 -66.75 Glyc3P+NAD+Pi+ADP 3PG+ATP+NADH -6.5 -0.56 -33.5 -69.06 3PG 2PG 2.1 -0.27 -24.7 -70.17 PG2 PEP -1.3 -0.64 -30.2 -72.88 PEP+ADP PYR+ATP -12.2 -7.4 -81.2 -103.89 PYR+NADH Lac+NAD -11.9 0 -131.0 -103.8
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My version of fig. 11.11
Data from Hamori (1975), J.Chem.Ed.52:370
Standard and actual free energy
-140
-120
-100
-80
-60
-40
-20
0
0 1 2 3 4 5 6 7 8 9 10
Step in glycolysis
Cumulative free energy changes, kJ mol-1
Cum DGo'
Sum DG
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Which steps are irreversible? Just three:
Glucose to G-6P (G ~ -40 kJ mol-1) Fructose-6-P to Fructose-1,6-bisP (-26) PEP to pyruvate (-31)
All the others are reversible So the controls are likely to be at those
three points: and they are!
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Regulation of glycolysis Two ways to study this:
Enzymology (know thy enzymes) Metabolic biochemistry (know concentrations
and fluxes under cellular conditions)
Sometimes enzymology gives interesting but cellularly unrealistic results (e.g., inhibitors that only inhibit at 100 * actual cellular concentrations)
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Regulators of glycolysis
See fig. 11.12: Glucose-6-P inhibits hexokinase ATP and citrate inhibit PFK-1 AMP, Fructose 2,6-bisP activate PFK1 F 1,6-bisP activates pyruvate kinase ATP inhibits pyruvate kinase
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Control at the transport level [glucoseintracellular] < [glucoseblood]
(except in liver);passive transport aided by transporters
All mammalian cells have transporters Na+ dependent cotransport: SGLT1
in intestinal & kidney cells GLUT family (1-7) found in other cells
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Insulin and Glut4 (fig. 11.13) When insulin binds to tyr-kinase receptors, they
dimerize and promote fusion of intracellular vesicles with the plasma membrane
Vesicles carry Glut4 transporters This happens only in striated muscle and
adipose tissue—that’s where the Glut4 transporters are
This is only one of several roles that insulin plays in glucose and lipid metabolism
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How does glucose get in? SGLT1 and GLUT4 stories (above) GLUT1,3 provide basal intake levels GLUT2 brings glucose in & out of liver GLUT5: fructose in small intestine GLUT7: G6P from cytoplasm to ER Doesn’t stay neutral long:
once it gets into the cell, it gets 6-phosphorylated with help of hexokinase
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Regulation of hexokinase Isozymes I,II,III: Km ~ 0.1mM;
G6P allosterically inhibits the enzyme Glucokinase (IV): unregulated, high Km
… found in liver & islet cells Pileup of G6P occurs if downstream
steps are inhibited;allostery in hexokinase I-III alleviates that
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GKRP and F-6P: regulators of liver glucokinase
Glucokinase regulatory protein binds glucokinase in presence of F-6-P and F-1-P Lowers affinity to ~ 10mM sigmoidal kinetics
With high [glucose], GKRP pulls GK into nucleus; low [glucose] makes GKRP release GK so it can phosphorylate glucose
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Regulation of PFK-1 Nucleotides:
ATP is both substrate and(usually) allosteric inhibitor
ATP increases apparent Km for F6P AMP is activator: relieves ATP inhibition ADP’s effects vary [ATP] fairly constant; [AMP] varies
Citrate (Krebs cycle component) inhibits it [H+] is also an inhibitor (lactic acid debt)
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F-2,6-bisP and PFK-1, PFK-2 Potent activator of PFK-1 Absent in prokaryotes F-2,6-bisP Formed by action of PFK-2
ATP + F-6-P F-2,6-bisP + ADP Stimulated by Pi, inhibited by citrate Same enzyme is also fructose 2,6-
bisphosphatase at different active site See fig. 11.16!
Fructose 2,6-bisphosphate(n.b.: drawn backward from text)
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PFK-2 and glucagon High [glucagon] turns on adenylyl
cyclase pathway in liver Protein kinase A then
phosphorylates a serine in PFK-2 That turns on phosphatase activity,
turns off PFK-2 activity Thus [F-2,6-bisP] , PFK-1 less
active, glycolysis is depressedPhosphofructokinase-2PDB 2AXN57 kDa monomer
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What if glucose is being rapidly metabolized?
[glucagon] , [F-6-P], [F-2,6-bisP] F-6-P is a substrate for PFK-2 F-6-P is a potent inhibitor of F-2,6-
bisphosphatase
That activates a phosphatase that dephosphorylates PFK-2
PFK-2 activity , phosphatase activity ! See figure 11.17
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Pyruvate kinase regulation Four isozymes in mammals Liver, kidney, blood forms have sigmoidal
kinetics for [PEP] Activated by F-1,6-bisP, inhibited by ATP
Low [F-1,6-bisP]:ATP almost completely inhibits enzyme
High [F-1,6-bisP]: ATP almost irrelevant Feed-forward activation
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Pyruvate kinase, phosphorylation, and glucagon
One isozyme (liver, intestine) is sensitive to [glucagon]:
Protein kinase A (see PFK-2!) phosphorylates pyruvate kinase, inactivating it somewhat
Glucagon stimulates protein kinase A, so it tends to inactivate pyruvate kinase
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Pasteur effect
Definition: increase in glycolysis under anaerobic conditions Relevant to yeast behavior Also to muscle metabolism when exercising,
since not enough [O2] is getting to the muscles to maintain oxidative phosphorylation
Reason: less ATP per glucose molecule with anaerobic metabolism, so you need to use more glucose to get the same amount of ATP out
Modulation at PFK-1 level, others
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Fructose Transported with GLUT5 Ordinarily phosphorylated to F-1-P by ATP-
dependent fructokinase F-1-P cleaved to DHAP and glyceraldehyde
by fructose 1-P aldolase Glyceraldehyde is 3-phosphorylated by
ATP-dependent triose kinase DHAP, Glyc-3-P then enter glycolysis as
usual
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Fructose-metabolizing enzymes
Fructokinase F-1P aldolase
(now considered a subset of ordinary F-1,6-bisP aldolase)
Triose kinase (no structures yet!)
FructokinasePDB 2hlzhuman136 kDa tetramer