Molecular Biochemistry I

8/3/2019 Molecular Biochemistry I

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Molecular Biochemistry I

Glycolysis and Fermentation

Contents of this page:

Glycolysis pathway reactionsSummary of pathwayFermentationRegulation of glycolysis

Glycolysis PathwayThe Glycolysis pathway is described below and summarized in Fig. 17.3 p. 584 ofBiochemistry, by Voet & Voet, 3rd Edition.

The reactions of Glycolysis take place in the cytosol of cells.

Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Initially,there is energy input corresponding to cleavage of two ~P bonds of ATP.

1. Hexokinase catalyzes: glucose+ ATP The Hexokinase reactioninvolves nucleophilic attack ofthe C6 hydroxyl oxygen ofglucose on the phosphorous ofthe terminal phosphate of ATP.

ATP binds to the enzyme as acomplex with Mg++.

The positively charged Mg++

interacts with negatively chargedphosphate oxygen atoms of ATP,providing charge compensation andpromoting a favorable conformationof ATP at the active site. (See alsodiagram p. 585.)

The reaction catalyzed byHexokinase is highly spontaneous.A phosphoanhydride bond of ATP(~P) is cleaved. The phosphate esterformed in glucose-6-phosphate hasa lower G of hydrolysis.
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Induced fit: Glucose binding to Hexokinase stabilizesa conformation in which

The C6 hydroxyl of the bound glucose is closeto the terminal phosphate of ATP, promoting

catalysis. Water is excluded from the active site. Thisprevents the enzyme from catalyzing ATPhydrolysis, rather than transfer of phosphate toglucose.

It is a common motiffor an enzyme active site to belocated at an interface between protein domains that areconnected by a flexible hinge region. The structuralflexibility allows access to the active site, whilepermitting precise positioning of active site residues,

and in some cases exclusion of water, as substratebinding promotes a particular conformation.

The Phosphoglucose Isomerasemechanism involves acid/basecatalysis, with ring opening,

isomerization via an enediolateintermediate, and then ringclosure (diagram p. 587). Asimilar reaction catalyzed byTriose Phosphate Isomerase ispresented in more detail below.


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This highly spontaneousreaction has a mechanismsimilar to that ofHexokinase.

The Phosphofructokinasereaction is the rate-limiting step ofGlycolysis. The enzymeis highly regulated, aswill be discussed later.

The Aldolase reaction is analdol cleavage, the reverse ofan aldol condensation.

Note that carbon atoms arerenumbered in reactionproducts.

A lysine residue at the active site ofthe Aldolase enzyme functions incatalysis.

The keto group of fructose-1,6-bisPreacts with the -amino group of theactive sitelysine, to form aprotonated Schiff base intermediate.

Cleavage of the bond between C3 andC4 follows. (See p. 590).


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The ketose/aldose conversion ofTIM involves acid/base catalysis,and is thought to proceed via anenediol intermediate, as withPhosphoglucose Isomerase,

Active site Glu and His residuesare thought to extract and donateprotons during catalysis.

2-Phosphoglycolate is an example of a transitionstate analog that binds tightly at the active site ofTriose Phosphate Isomerase. This inhibitor ofcatalysis by TIM is similar in structure to theproposed enediolate intermediate.

TIM is considered a "perfect enzyme", because thereaction rate is limited only by the rate at whichsubstrate collides with the enzyme.

The structure of Triose Phosphate Isomerase is an barrel, orTIM barrel.In an barrel there are 8 parallel -strandssurrounded by 8 -helices. Short loops connectalternating -strands and -helices.

TIM barrels serve as scaffolds for active site residues in adiverse array of enzymes. Residues that form the activesite are always located at the same end of the barrel,associated with the C-terminal ends of -strands and the

loops connecting these to -helices.

There is debate over whether the many different TIMbarrel enzymes are evolutionarily related, since in spite ofthe structural similarities there is tremendous diversity incatalytic functions of these enzymes and little sequencehomology.Explore at right the structure of the Triosephosphate


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Isomerase (TIM) homodimer, with the transition stateinhibitor 2-phosphoglycolatebound to one of the TIMmonomers.

Note the structure of the TIM barrel, and the loop thatforms a lid that closes over the active site after binding ofthe substrate.

Triose Phosphate Isomerase

Exergonic oxidation of the aldehyde inglyceraldehyde-3-phosphate, to acarboxylic acid, drives formation of an acylphosphate, a "high energy" bond (~P), in1,3-bisphosphoglycerate.

This is the only step in Glycolysis in which

NAD

+

is reduced to NADH.A cysteine thiolat the active site ofGlyceraldehyde-3-phosphateDehydrogenase has a role in catalysis (p.596).

The aldehyde of glyceraldehyde-3-phosphate reacts with the active sitecysteine thiol to form a thiohemiacetalintermediate. Oxidation to a carboxylicacid (in a "high energy" thioester) occurs,
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as NAD+ is reduced to NADH.

The "high energy" acyl thioester isattacked by Pi to yield the acyl phosphate

(~P) product.

Recall that NAD+accepts 2 e- plus one H+ (a hydride)in going to its reduced form.

This transfer of phosphate to ADP, from thecarboxyl group on 1,3-bisphosphoglycerate, isreversible (low G), since one ~P bond iscleaved and another is synthesized. Theenzyme undergoes a substrate-inducedconformational change similar to that ofHexokinase.

Phosphate is shifted from the hydroxyl on C3of 3-phosphoglycerate to the hydroxyl on C2.


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An active site histidine side-chainparticipates in phosphate transfer, bydonating and accepting the phosphate. The

process involves a 2,3-bisphosphateintermediate.

The Phosphoglycerate Mutase reaction is illustrated in theanimation at right.

of PhosphoglycerateMutase

This dehydration reactionis Mg++-dependent.

2 Mg++ ions interact withoxygen atoms of thesubstrate carboxyl groupat the active site. The Mg++ ions help to stabilize theenolate anion intermediatethat forms when a lysineside-chain amino groupextracts a proton fromcarbon #2.
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This transfer of phosphate from PEPto ADP is spontaneous. PEP has alarger G of phosphate hydrolysisthan ATP, because removal ofphosphate from PEP yields an

unstable enol, that spontaneouslyconverts to the keto form of pyruvate(p. 602). Required inorganic cationsK+ and Mg++ bind to anionic residuesat the active site of Pyruvate Kinase.Summary of Glycolysis:

The pathway continues fromglyceraldehyde-3-phosphate. Recallthat there are two glyceraldehyde-3-phosphate per glucose metabolized.

Balance sheet for high energy bonds ofATP:

2 ATP expended 4 ATP produced (2 from each of

two 3C fragments fromglucose)

Net production of 2 ~P bondsof ATP per glucose.


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For example, Lactate Dehydrogenase catalyzesreduction of the keto group in pyruvate to ahydroxyl, yielding lactate, as NADH is oxidized toNAD+.

Lactate, in addition to being an end-product offermentation, serves as a mobile form ofnutrientenergy, and possibly as a signal molecule inmammalian organisms. Cell membranes containcarrier proteins that facilitate transport of lactate.

Skeletal muscles ferment glucose to lactateduring exercise, when the exertion is briefand intense. Lactate released to the bloodmay be taken up by other tissues, or byskeletal muscle after exercise, and convertedvia Lactate Dehydrogenase back topyruvate, which may be oxidized inKrebs
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Cycle or (in liver) converted to back toglucose via gluconeogenesis.

Lactate serves as a fuel source forcardiac

muscle as well as brain neurons.Astrocytes, which surround and protectneurons in the brain, ferment glucose tolactate and release it. Lactate taken up byadjacent neurons is converted to pyruvatethat is oxidized via Krebs Cycle.

Some anaerobic organismsmetabolize pyruvate to ethanol,which is excreted as a wasteproduct. NADH is converted toNAD+ in the reaction catalyzed by

Alcohol Dehydrogenase.Thiamine pyrophosphate, thecofactor for AlcoholDehydrogenase, is discussedelsewhere.

Glycolysis Enzyme * G

o'(kJ/mol)

G(kJ/mol)

Hexokinase -20.9 -27.2

Phosphoglucose Isomerase +2.2 -1.4Phosphofructokinase -17.2 -25.9

Aldolase +22.8 -5.9

Triosephosphate Isomerase +7.9 negative

Glyceraldehyde-3-phosphate Dehydrogenase, & PhosphoglycerateKinase

-16.7 -1.1
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Phosphoglycerate Mutase +4.7 -0.6

Enolase -3.2 -2.4

Pyruvate Kinase -23.0 -13.9
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Glucokinase, with its high KM forglucose, allows the liver to storeglucose as glycogen in the fed statewhen blood [glucose] is high.

The liver enzyme Glucose-6-phosphatase catalyzes hydrolyticrelease of Pi from glucose-6-phosphate. Thus glucose is releasedfrom the liver to the blood as

needed to maintain blood [glucose].

The enzymes Glucokinase andGlucose-6-phosphatase, both foundin liver but not in most other bodycells, allow the liver to controlblood [glucose].


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Phosphofructokinase is usually the rate-limiting step of the Glycolysis pathway.Phosphofructokinase catalyzes:fructose-6-phosphate + ATP
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1. Explore in the Biochemistry Simulations tutorials at rightconcepts of enzyme kinetics and enzyme regulation relevantto this class:

In the module on Glucokinase, compare the

dependence on [glucose] of catalysis by Glucokinaseand Hexokinase.

In the module on Phosphofructokinase,explore effects of varied [ATP] at zero [fructose-2,6-bisphosphate]. (The activator fructose-2,6-bisphosphate will be discussed in the section ongluconeogenesis.)

Note: Hold down theControlkey while

clicking on the aboveicon.

Additionalmaterial

onGlycolysis:Readings,

TestQuestions,& Tutorial
http://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/gluconeo.htm#regulationhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/gluconeo.htm#regulationhttp://www.terravivida.com/vivida/diygly/http://www.terravivida.com/vivida/diygly/http://www.terravivida.com/vivida/diygly/http://www.terravivida.com/vivida/diygly/http://www.terravivida.com/vivida/diygly/http://www.terravivida.com/vivida/diygly/http://www.terravivida.com/vivida/glysteps/http://www.terravivida.com/vivida/glysteps/http://www.terravivida.com/vivida/glysteps/http://www.terravivida.com/vivida/glysteps/http://www.terravivida.com/vivida/glysteps/http://www.terravivida.com/vivida/glysteps/http://www.terravivida.com/vivida/glysteps/http://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/tq-glycol.htmhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/tq-glycol.htmhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/tq-glycol.htmhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/tq-glycol.htmhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/indexlist.htmlhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/MB1index.htmlhttp://www.rpi.edu/dept/bio/info/BiochemModules/Launcher.htmlhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/gluconeo.htm#regulationhttp://www.terravivida.com/vivida/diygly/http://www.terravivida.com/vivida/diygly/http://www.terravivida.com/vivida/glysteps/http://www.terravivida.com/vivida/glysteps/http://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/tq-glycol.htmhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/tq-glycol.htmhttp://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/tq-glycol.htm

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Molecular Biochemistry I