§ 3.4 Protein Catabolism - PowerPoint Presentation

Slide1

§3.4 Protein Catabolism §3.4a Protein Degradation §3.4b Amino Acid Breakdown §3.4c Urea Cycle

III. METABOLIC BIOCHEMISTRY

Slide2

§3.4a Protein Degradation

Slide3

Synopsis 3.4a

Dietary

proteins are degraded

into free amino acids by

the

collaborative

action

of digestive proteases

The bulk of free amino acids

released from the breakdown of dietary proteins is funneled toward the synthesis of cellular proteins

However, excess amino acids are usually converted to glucose, acetyl-CoA, and ketone bodies—and thus serve as

metabolic fuels

During

starvation

, the degradation of cellular proteins within

tissues also serves

as an alternative source of free amino acids that are ultimately broken down into metabolic intermediates for energy

production—such

degradation usually occurs within

lysosomes

Slide4

In addition to pepsin, dietary proteins are degraded into free amino acids by the collaborative action of three other major digestive proteases: trypsin, chymotrypsin, and elastaseWith preference for hydrophobic and aromatic residues, pepsin displays a

rather high

degree of promiscuity (or broad specificity) in its ability to cleave peptide bonds

Dietary Protein Degradation

—

Ala—

Ser

—

Phe

—

Ser—Lys—Gly—Ala—Arg—Trp—Thr—Asp—Tyr—Gly—Lys—Cys—

Trypsin

Chymotrypsin

Elastase

Slide5

Within cells, proteins are constantly turned over—ie proteins typically have half-lives ranging from minutes to days (and weeks or more in some cases)—regulatory proteins such as transcription factors usually have a high turn over During starvation, the degradation of cellular proteins within tissues—such as liver, kidney, and skeletal muscle—serves as an alternative source of free amino acids that are ultimately broken down into metabolic intermediates for energy production Such degradation usually occurs within lysosomes

—that harbor selective importers and hydrolytic proteases such as

cathepsins

for the breakdown of cytosolic proteins into free amino acids in a manner akin to the action of digestive proteases

Cellular Protein Degradation

Degradation

Protein

Free

Amino Acids

Slide6

The released amino acids from the degradation of cellular and dietary proteins enter the bloodstream, the latter through the digestive tract (small intestine) via a number of amino acid transporters—and are subsequently transported to hepatocytes in the liver Once inside

hepatocytes, excess dietary

amino

acids are broken down into metabolic intermediates

, many of which enter

the Krebs cycle

for energy

production

Amino Acid Absorption

Intestinal Lumen

Brush Border CellBlood Capillary

Slide7

Exercise 3.4a

Describe

how dietary proteins are broken down

Compare the substrate specificities of trypsin and chymotrypsin

What are the major organs where cellular

proteins are broken

down during times of starvation?

After their release from dietary proteins, how are free amino acids absorbed into the bloodstream?

Slide8

§3.4b Amino Acid Breakdown

Slide9

Synopsis 3.4b

After their release from dietary/cellular proteins,

free amino acids can be broken down into the following metabolites

for energy production (or in biosynthetic pathways):

α

-

Ketoglutarate

Succinyl

-CoA

Fumarate

OxaloacetatePyruvateAcetoacetateAcetyl-CoAOf these seven metabolites, four are Krebs cycle intermediates: - -ketoglutarate - Succinyl-CoA

-

Fumarate

- Oxaloacetate

Pyruvate

and acetoacetate (a ketone body) can be easily converted to acetyl-CoA–the spark that starts the Krebs cycle “ignition” by virtue of its ability to donate a two-carbon unit in the form of an acetyl groupIn a nutshell,

the breakdown products of amino acids essentially serve as a “fuel

” for the Krebs cycle—but be aware that a

cetyl-CoA can also be converted into fatty acids

!

Slide10

Amino Acid Breakdown: Glucogenic and Ketogenic Precursors

In the context of their catabolic breakdown, amino acids can be divided into two groups:

Glucogenic

amino acids

—

these are amino acids that can be directly broken

down into glucose precursors such as pyruvate,

-

ketoglutarate

,

succinyl-CoA, fumarate, or oxaloacetate—used in the synthesis of glucose (gluconeogenesis)Ketogenic amino acids—these are amino acids that can be directly broken down into ketogenic precursors such as acetyl-CoA or acetoacetate—used in the synthesis of ketone bodies (ketogenesis)

Helpful mnemonics:

Ketogenic (2)

 KL

Glucogenic (13) 

CHARMED-GNQ-SVPKetogenic/Glucogenic (5)  WIFTY

Slide11

Amino Acid Breakdown: Transamination and DeaminationNH3 + H2O <==> NH

4

+

+ HO

-

pKa

 9@ pH =

7.4

=> NH3 = NH4+Under physiological settings, NH3 largely exists as NH4+Two major mechanisms involved in amino acid breakdown are:1) Transamination (cytosolic)2) Deamination (mitochondrial)

Slide12

1:1Formate(Methanoate)-2:1Acetate(Ethanoate)-

3:1

Propionate

(

Propanoate

)

-

4:1

Butyrate(

Butanoate

)-5:1Valerate(Pentanoate)-2:2Oxalate(

Ethanedioate

)-

-

3:2Malonate

(Propanedioate)--

4:2Succinate(

Butanedioate)

-

-5:2Glutarate(Pentanedioate

)

--

Monocarboxylate

Anions

Dicarboxylate

Dianions

6:1Caproate(Hexanoate)

-

Amino Acid Breakdown:

Relationship to Mono- and Dicarboxylic Acids

Slide13

(-Aminoglutarate)Aminotransferase

1) Transamination: General Features

In transamination

, the —NH

2

group of an

amino acid (the donor)

is transferred to an

-

keto

acid (the acceptor)—the most common -keto acid acceptor is -ketoglutarateCatalyzed by aminotransferase

or

transaminase (specific for each amino acid), t

he transamination reaction of an amino acid with -

ketoglutarate produces glutamate and the -

keto acid of the original amino acid—which is either a simpler metabolite or ultimately converted to one for subsequent oxidation to produce energyOther than -ketoglutarate,

oxaloacetate (-

ketosuccinate) and

pyruvate (

-ketopropionate) also serve as important -keto acid acceptors in the context of amino acid transamination

Transamination of amino acids occurs in the cytosol

of not only livers cells but also

peripheral tissues such as the heart muscle, skeletal muscle, and kidneys

Slide14

Glutamate—the end-product resulting from the transamination of most amino acids—often donates its —NH2 group to oxaloacetate (-ketosuccinate) to regenerate -

ketoglutarate

(particularly in liver cells)

Such transamination reaction is catalyzed by

aspartate aminotransferase

, producing

aspartate as a by-product (particularly in liver cells)—or the reaction may proceed in the reverse direction to produce glutamate so as to remove the amino group of other amino acids (particularly in peripheral tissues)In liver cells,

aspartate

serves as a key metabolite in the urea cycle (next section)1) Transamination: Regenerating -Ketoglutarate (reversible)

Aspartate

CH

2



OOC —Aspartate

Aminotransferase

Oxaloacetate

CH

2



OOC —

(

-

Aminoglutarate)

(

-Aminosuccinate)(-Ketosuccinate)

Liver Cells

Peripheral Tissues

Slide15

In peripheral tissues, glutamate—the end-product resulting from the transamination of most amino acids—is ultimately converted to alanine (through the alanine cycle)Catalyzed by alanine aminotransferase, the transfer of –NH2

group of

glutamate to pyruvate generates α-

ketoglutarate

and

alanine

Alanine

enters the bloodstream and is transported to liver cells—alanine

thus serves as a nitrogen-carrier between peripheral tissues and liver

In liver cells, the build up of alanine drives the equilibrium in favor of glutamate (the above reaction reverses), which will be ultimately deaminated to NH3 Pyruvate meets metabolic fates such as the Krebs cycle or gluconeogenesis

1) Transamination: Glutamate to Alanine (reversible)

Alanine

Aminotransferase

Alanine

CH

3

Pyruvate

CH

3

(

-

Aminoglutarate

)

(

-

Ketopropionate)(-

Aminopropionate)

Liver Cells

Peripheral Tissues

Slide16

In peripheral tissues, glutamate—the end-product resulting from the transamination of most amino acids—can also be converted to glutamine through condensation with NH4+ by glutamine synthetase, in what can be envisioned as a

pseudo-transamination

reaction

Glutamine

enters the bloodstream and is transported to liver cells—

glutamine

thus also serves as a nitrogen-carrier between peripheral tissues and liver

In liver cells, glutamine is partially

deaminated to glutamate—which is further deaminated to -ketoglutarate

to

completely eliminate NH4+ (vide infra)Together with alanine, glutamine thus plays a key role in the transport of nitrogen from amino acids in peripheral tissues to liverGlutamineSynthetaseATP + NH4+

ADP + P

i

O

NH3 H2

N—C—CH2

—CH2—CH—COO



+Glutamine

Glutamate

O

NH3

O—C—CH2—CH2—CH—COO

+

1) Transamination: Glutamate to Glutamine (irreversible)

Slide17

In deamination, the —NH2 group is removed in the form of NH4+ from an amino acid by a group of enzymes called deaminases—the corresponding

-

keto

acid

is

either a simpler metabolite or ultimately converted to one for subsequent oxidation to produce

energy

Deamination of glutamate (and most other amino acids) primarily occurs within the mitochondrial

matrix

of liver cells (and kidney cells to a lesser extent) Such compartmentalization of deamination within the mitochondrial matrix limits the toxic effect of NH4+—prior to its detoxification via the urea cycle (next section)After their transport from peripheral tissues into liver cells, the nitrogen-carriers alanine and glutamine are converted back to

glutamate

, which is subsequently deaminated into

-ketoglutarate and NH

4+

In liver cells, alanine undergoes transamination with -ketoglutarate to generate glutamate in a reverse reaction driven by

alanine aminotransferase (vide supra)On the other hand, the liver

glutaminase catalyzes direct deamination of the sidechain —NH

2

group of glutamine to generate glutamate (vide infra)

2

) Deamination: General Features

Deaminase

H

2

O

NH

4

+

Slide18

Glutamine—largely from peripheral tissues as a nitrogen-carrier—can be partially deaminated into glutamate and NH4+ within the mitochondrial matrix of liver cells

Reaction catalyzed by

glutaminase

— using H

2

O as a nucleophile to eliminate NH

4

+

Glutamate can be further deaminated

by glutamate dehydrogenase (vide supra)

NH4+ enters the urea cycle (next section) GlutaminaseNH4+

O

NH3

H2N—

C—CH2—CH2—CH—COO

+Glutamine

H

2

O

Glutamate

O

NH3

O—C—CH2—CH2—CH—COO

+

2

) Deamination: Glutamine



Glutamate

(irreversible

)

Slide19

Within the mitochondrial matrix of liver cells, glutamate—the end-product resulting from the transamination of most amino acids—is subsequently oxidized by glutamate dehydrogenase to α-ketoglutarate

with concomitant release of

NH

4

+

in a reaction termed “

oxidative

deamination”Glutamate dehydrogenase utilizes NAD

+

or NADP+ as an oxidizing agent Deamination proceeds by dehydrogenation of the C-N bond followed by hydrolysis of the resulting Schiff base (an imine harboring C=N bond)-Ketoglutarate meets metabolic fates such as the Krebs cycleNH

4

+ enters the

urea cycle (next section)

Glutamate Dehydrogenase

2) Deamination: Glutamate 

-Ketoglutarate

(irreversible)

(

-Aminoglutarate)

Slide20

Asparagine can be partially deaminated into aspartate and NH4+ within mitochondrial matrix of liver cells

Reaction catalyzed by

asparaginase

—using

H

2

O as a nucleophile to eliminate NH

4

+Aspartate can undergo transamination to produce oxaloacetate (vide supra), or enter the

urea cycle (next section)

NH4+ enters the urea cycle (next section)AsparaginaseNH4+ O

NH3

H

2N—C—CH

2—CH—COO +Asparagine

H

2

O

Aspartate

O

NH3

O

—C—CH

2—CH—COO +

2

) Deamination: Asparagine



Aspartate

(irreversible

)

Slide21

Like serine, threonine can be directly deaminated into -ketobutyrate and NH4+ within the mitochondrial matrix of liver cells

Reaction

catalyzed by

threonine dehydratase

—using

H

2

O as a nucleophile to eliminate NH

4+-

Ketobutyrate

usually enters Krebs cycle after its conversion into succinyl-CoANH4+ enters the urea cycle (next section)+ThreonineDehydratase

NH

4+

OH

NH3

H3C—CH—CH—COO

Threonine

-Ketobutyrate

H

2O

O

H

3C—CH2

—C—COO

2

) Deamination: Threonine



-

Ketobutyrate

(irreversible)

Slide22

Unlike the transamination of most amino acids into glutamate, serine (as well as alanine and cysteine) can be directly deaminated into pyruvate and NH4+—these reactions largely occur within the cytosol of liver cells (possibly due to a major role of pyruvate in gluconeogenesis)

Serine deamination is catalyzed by

serine dehydratase

—using H

2

O as a nucleophile to eliminate NH

4

+

Pyruvate usually enters Krebs cycle or gluconeogenesis NH4

+

enters the urea cycle (next section)SerineDehydrataseNH4+ NH3

HO—CH

2—CH—COO



+

SerinePyruvate

H

2O

O

CH3—C—COO

2

) Deamination: Serine



Pyruvate

(irreversible

)

Slide23

Exercise 3.4b

Describe the two general metabolic fates of the carbon skeletons of amino acids

List the seven metabolites that represent the end products of amino acid catabolism. Which are

glucogenic

? Which are ketogenic?

Which amino acids can be broken down into

the Krebs cycle

intermediates?

Which amino acids can be broken down into pyruvate?

Which amino acids can be broken down into acetyl-CoA and/or acetoacetate?

Slide24

§3.4c Urea CycleCamels can go without water for months!

Slide25

Synopsis 3.4c

Excess

nitrogen (the –NH

2

group)

resulting from

the breakdown of free amino acids into metabolic fuels is released

in the form of NH

3

(strictly NH4+)Where water is plentiful, many aquatic animals directly excrete NH4+ in the urine In terrestrial vertebrates, NH4+ is converted to less toxic urea—primarily in the liver

but also in kidneys to a lesser extent—via the so-called urea cycle

After its synthesis in the liver,

urea is secreted into the bloodstream and ultimately sequestered by the kidneys for excretion in the urine

Reaction of NH4+ (NH3 originating from amino acid breakdown) with HCO3

- (

eg CO2

from tissue respiration and decarboxylation) produces

urea via the following reaction: NH3 + CO2

+ H2

O + aspartate + 3ATP + H

2O

NH4+

+ HCO3 + aspartate + 3ATP + H

2O

urea

+ fumarate + 2ADP + AMP +

PPi + 2P

i

Urea

(carbamide)

Slide26

Overall Reaction

Urea’s two –NH

2

groups are derived from

NH

4

+

(ultimately from amino acid breakdown) and

aspartate (an amino acid), while the central C=O group hails from the HCO3-

H

2ONH4+

Slide27

The Urea Cycle—a largely liver affair!

First ever metabolic cycle

discovered by Krebs and

Henseleit

in 1932

It is called “

cycle

” rather than a “

pathway

” because it cycles ornithine back to itself—

ie the substrate and the product are identical! Comprised of five enzymatically-driven metabolic steps (Steps 1-5)—the first two steps occur within the mitochondrial matrix, while latter three in the cytosol of liver cellsAlthough essential,

Steps 1

is technically not an integral component of the urea

cycle

Of the four metabolic intermediates of urea cycle, three are non-standard or

non-proteinogenic amino acids!

Slide28

Urea Cycle: 1 Carbamoyl Phosphate Synthetase I (CPS-I)

In the mitochondrial matrix,

NH

4

+

resulting from the deamination of amino acids is condensed with

HCO

3

- (eg

from tissue respiration and

decarboxylation) to generate carbamoyl phosphate—cf similarity with urea H2N—C(O)—NH2 Reaction is catalyzed by CPS-I and powered by ATPCPS-I (mitochondrial matrix) is one of the two major forms of CPS—CPS-II (cytosolic) uses glutamine as a source of nitrogen to generate carbamoyl phosphate involved in the biosynthesis of pyrimidine nucleotides (see

§

4.1)

Carbamoyl phosphate is a carboxamide

with the formula R—C(O)NH2

, the functional group of which is referred to as CARBAMOYL (prefix) or AMIDE (suffix)—eg carbamoyl phosphate may also be written as phospoamide!

Carbamoyl phosphate is an activated molecule (cf UDP-glucose in

§3.2) in that it can readily donate its

carbamoyl moiety —C(O)NH

2 to a substrate–enter ornithine O

NH

4+ +

HCO3-

+ 2ATP

H2N—

C—

OPO32- + 2ADP + Pi CPS-I

Carbamoyl

phosphate

Slide29

Urea Cycle: 2 Ornithine Transcarbamoylase (OTC)

In the mitochondrial matrix, the

carbamoyl moiety —C(O)NH

2

of carbamoyl phosphate is transferred to

ornithine (

cf

lysine) to generate citrulline (carbamoyl ornithine!)

—both of which are non-standard amino acids in that they play no role in protein biosynthesis

Reaction is catalyzed by OTC producing inorganic phosphate (Pi) as a by-productBoth ornithine (produced in the cytosol) and citrulline (produced in the mitochondrion) require specific transporters located within the inner mitochondrial membrane (IMM) for their transport in and out of mitochondria—the next three steps of urea cycle all occur within the cytosol ending with the recycling of ornithine + O NH3

H

2N—C—

OPO32-

+ H3N—(CH2)3—CH—COO- OTC

O

NH

3

H2N—

C—HN—(CH2

)3—CH—COO

-

P

i

Carbamoyl

phosphate

Ornithine

(

-

Aminopropyl

-glycine

)

Citrulline(

Carbamoyl ornithine)+

+

Slide30

Urea Cycle: 3 Argininosuccinate Synthetase (ASS) COO-

-

OOC—CH

2

—CH—NH

3

ASS

O

NH3 H2N—C—HN—(CH2)3—CH—COO-

AMP +

PP

i

Aspartate

(-Carboxymethyl-glycine)++

COO-

NH2

NH3 -OOC—CH2—CH—NH—

C—HN—(CH2

)3—CH—COO

-

Argininosuccinate

(

Succinylarginine

)++

ATP

+

In the cytosol,

aspartate

is condensed with

citrulline

to generate

argininosuccinate

(

succinylarginine

!)

—the third non-standard amino acid in the urea cycle

Reaction is catalyzed by

ASS

in the presence of ATP, producing AMP and pyrophosphate (

PP

i

) as by-products—the spontaneous hydrolysis of the latter drives the forward reaction

Of the two —NH

2

groups in urea

H

2

N—

C(O)

—

NH

2

, one is supplied by

NH

4

+

(resulting from the deamination of glutamate) and the other by

aspartate

Citrulline

(

Carbamoyl ornithine

)

Slide31

Urea Cycle: 4 Argininosuccinase (ASL) COO- -

OOC—HC—CH

ASL

NH

2

NH

3

H2N—C—HN—(CH2)3—CH—COO- Fumarate(Didehydrosuccinate

)

+

+

COO-

NH2 NH3 -

OOC—CH2

—CH—NH—C—HN—(CH

2

)3—CH—COO-

+

+

Arginine

(

-Guanidinopropyl-

glycine

)+

In the cytosol,

argininosuccinate

is cleaved (or split up) into

fumarate

and arginine

Reaction is catalyzed by ASL (

argininosuccinase or argininosuccinate

lyase

)—recall that lyase

breaks chemical bonds by means other than hydrolysis

Fumarate is ultimately converted to oxaloacetate by cytosolic enzymes in a manner akin to its fate in the Krebs cycle (see §

3.5)—oxaloacetate usually enters gluconeogenesis

Arginine

continues to travel along the urea cycle as it serves as the precursor of urea

Argininosuccinate

(

Succinylarginine

)

Slide32

Urea Cycle: 5 Arginase (ARG) ARG NH3

H

3

N—(CH

2

)3—CH—COO-

+

H2O+ NH2 NH3 H2N—C—HN—(CH2

)

3—CH—COO-

+

+ O

H2N—

C

—NH2Urea(

Carbamide)+

In the cytosol, arginine is hydrolyzed into

ornithine

and

urea

Reaction is catalyzed by

ARG

using H

2O as a nucleophile to eliminate urea—

ie arginase is an hydrolase!

Ornithine is shuttled back into the mitochondrial matrix for another round of detoxification of NH

4+

Urea

is secreted into the bloodstream and ultimately sequestered by the kidneys for excretion in the urine

Arginine

(-Guanidinopropyl

-glycine)

Ornithine(

-Aminopropyl

-glycine

)

Slide33

Exercise 3.4c

What is the difference between carbamoyl and amide functional groups?

Compare the chemical structures of carbamoyl phosphate and urea (carbamoyl amine)

Summarize various steps of the urea cycle