25 Mar 2008 Glycolysis Andy Howard Introductory Biochemistry 25 March 2008

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*


Glycolysis Now we’re ready for the specifics of

metabolism Why glycolysis first?

Well-understood (?) early on Illustrates concepts used later Inherently important


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


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


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


The pathway through TIM

Fig. courtesy U.Texas


Pathway to pyruvate

Bottom half of same graphic


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


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


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.


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


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


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


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


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


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


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.

+


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


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.


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


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


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.


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


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


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


Enolase interconverts 2-

phosphoglycerate & phosphoenolpyruvate

This reaction plays a role in gluconeogenesis as well as glycolysis.

PDB 4enlSaccharomyces97 kDa dimer; monomer shown


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.


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


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


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


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


Lactate dehydrogenase

Typical tetrameric Rossmann-fold NAD-dependent dehydrogenase

Structural homology to other NAD-binding enzymes

PDB 1xivPlasmodium140 kDa tetramer


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


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


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


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.


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


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


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!


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)


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


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


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


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


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


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


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)


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)


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


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


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


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


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


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


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

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25 Mar 2008 Glycolysis Andy Howard Introductory Biochemistry 25 March 2008