Chapter 23 - Protein Turnover and Amino Acid Metabolism

Amino acids are obtained from the diet when proteins are ______

digested

_____ ______ are degraded to amino acids because of damage, misfolding, or charging metabolic demands

cellular proteins

Excess amino acids cannot be stored or excreted, so they must be used as _____ _____

metabolic fuel

Dietary proteins are degraded to ____ _____ which are absorbed by the intestine and transported in the blood

amino acids

Essential amino acids

amino acids that cannot be synthesized and must be acquired in the diet

Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine

Essential amino acids

The digestion of dietary proteins begins in the _____ and is completed in the ______

stomach; intestine

Protein digestion begins in the ______, where the acidic environment denatures proteins into random coils

stomach

Pepsin

the primary proteolytic enzyme of the stomach (maximally active at pH 2)

Partly digested proteins move from the stomach to the beginning of the ____ _____ (duodenum), stimulating the secretion of sodium bicarbonate and proteolytic enzymes from the pancreas

small intestine

_________ in the plasma membrane of intestinal cells enhance digestion

Aminopeptidases

The products of protein digestion are absorbed by the _____ ______

small intestine

Free amino acids, dipeptides, and tripeptides are transported into the ______ _____

intestinal cells

At least ____ different transporters exist, each specific to a different group of amino acids

seven

Absorbed amino acids are released into the _____ by a number of Na+–amino acid antiporters.

blood

Protein turnover

the degradation and resynthesis of proteins

Protein turnover is essential for removing short-lived or ______ proteins

damaged

Which is an essential amino acid in

humans?

a. arginine

b. cysteine

c. lysine

d. proline

e. serine

c. lysine

Ubiquitin

a small (76 aa) protein that tags proteins for destruction

Ubiquitin is present in all _____ cells and requires ____ _____

eukaryotic; ATP hydrolysis

Ubiquitin attaches by its carboxyl-terminal ____ residue to the ε-amino groups of 1+ ____ residues on the target protein

Gly; Lys

Ubiquitin is a small, compact protein with seven ______

lysines

_____ enzymes participate in the attachment of ubiquitin to a protein

Three

adenylates ubiquitin and transfers it to a sulfhydryl group of a Cys residue of E1 (requires ATP)

Ubiquitin-activating enzyme (E1)

transfers ubiquitin to one of its own sulfhydryl groups

Ubiquitin-conjugating enzyme (E2)

transfers ubiquitin from E2 to an ε-amino group on the target protein

Ubiquitin–protein ligase (E3)

__ can remain bound to the target protein and generate a chain of ubiquitin molecules

E3

E3 can _____ after the first ubiquitin addition, and a chain can be extended by another E2/E3 pair.

dissociate

Ubiquitin can be added onto any of the seven Lys or the ______of the previous ubiquitin

N-terminus

A chain of 4+ ubiquitin molecules linked via Lys 48 is an especially effective signal for ____ ______

protein degradation

In a tetraubiquitin chain, four ubiquitin molecules are linked by _____ bonds

isopeptide

The ε-amino group of a Lys residue of one ubiquitin is linked to the terminal

_______ of another

carboxylate

Degron

a specific sequence of amino acids that indicates a protein should be degraded

For many proteins, the amino-terminal residue amino acid (N-degron) is an important degradation signal for ___ enzymes

E3

Other degrons include

cyclin destruction boxes and PEST sequences

Proteins that are not degraded because of a ____ ____ may accumulate, causing a disease of protein aggregation

defective E3

A severe neurological disorder characterized by an unusually happy disposition, cognitive disability, absence of speech, uncoordinated movement, and hyperactivity is called

Angelman syndrome

Angelman syndrome is caused by a ___ in a member of the __ family

defect; E3

Overexpression of an E3 causes _______

autism

Inappropriate protein turnover can lead to _____

cancer

The _____ digests the ubiquitin-tagged proteins

proteasome

Proteasome (26S proteasome)

a large, ATP-driven protease complex that digests ubiquitinated proteins

The 26S proteosome is a complex of two components:

– one ___ catalytic unit arranged as a barrel

– two ___ regulatory units that control access to the interior of the 20S catalytic subunit

20S; 19S

The 20S proteasome is barrel-shaped and made up of 28 homologous subunits

28

The 19S regulatory units:

– contain ubiquitin receptors that ____ specifically to polyubiquitin chains

– use ATP to ____ polyubiquitinated chains and direct them into the catalytic core

– contain an isopeptidase that ____ off intact ubiquitin molecules so that they can be reused

bind; unfold; cleaves

Key components of the 19S complex are ____ ATPases of the AAA+ class (ATPases associated with various cellular activities)

six

There are ____ types of active sites in the β subunits (barrel) , each with a different specificity

three

All active sites employ an N-terminal ____ residue

– The hydroxyl group of the Thr residue attacks the _____ groups of peptide bonds, forming acyl-enzyme intermediates

– Substrates are degraded in a processive manner without intermediate release

– Substrates are reduced to ____ ranging from seven to nine residues before release

Thr; carbonyl; peptides

The proteasome and other proteases generate ____ _____ _____

free amino acids

The ubiquitin pathway and the proteasome appear to be present in all ______

eukaryotes

Homologs of the proteasome are also found in some ______

prokaryotes

Proteasomes have become more _____ in eukaryotes

complex

In the archaeal proteasome, all α outer-ring subunits and all β inner-ring subunits are _______

identical

In eukaryotes, each α or β subunit is one of the seven ______ isoforms, which provides different substrate specificity

different

Bortezomib (Velcade)

a dipeptidyl boronic acid inhibitor of the proteasome

________ is used as a therapy for multiple myeloma

Bortezomib

Degrons are used as regulatory mechanisms for protein ________

expression

HT1171

a suicide inhibitor of the proteasome of M. tuberculosis (has no effect on human proteasomes)

The first step in amino acid degradation is the removal of ______

nitrogen

Amino acids not needed as building blocks are degraded to compounds able to enter the ______ mainstream

metabolic

For amino acids not needed, the _____ group is removed, and then the remaining ____ _____ is metabolized to a glycolytic intermediate or to acetyl CoA

amino; carbon skeleton

The major site of amino acid degradation in mammals is the _____

liver

Muscles also readily degrade the branched-chain amino

acids(____, ____, and _____).

Leu, Ile, and Val

The α-amino group is transferred to α-ketoglutarate, yielding _____

glutamate

Glutamate is oxidatively deaminated in the liver to yield ______ ion (NH4+)

ammonium

Aminotransferases (transaminases)

catalyze the

transfer of an α-amino group from an α-amino acid to an α-ketoacid (reactions are reversible and can be used to synthesize amino acids from α-ketoacids)

Aspartate aminotransferase

catalyzes the transfer of the amino group of aspartate to α-ketoglutarate

Alanine aminotransferase

catalyzes the transfer of the amino group of alanine to α-ketoglutarate

Glutamate dehydrogenase

a mitochondrial enzyme that converts the nitrogen atom in glutamate to a free ammonia ion by oxidative deamination

Glutamate dehydrogenase is a ___-specific enzyme

liver

Can use either NAD+ or NADP+

Glutamate dehydrogenase

Glutamate dehydrogenase proceeds by dehydrogenation of the C–N bond, followed by ______ of the ketimine

hydrolysis

Glutamate dehydrogenase is allosterically inhibited by ____ and stimulated by ADP in mammals

GTP

In most terrestrial vertebrates, NH4+ is converted into ____, which is excreted

urea

Aminotransferases require the coenzyme ____ ____ ____ (__), a derivative of pyridoxine (vitamin B6)

pyridoxal phosphate (PLP)

Pyridoxal phosphate derivatives can form a stable _____ form

tautomeric

In the absence of substrate, PLP forms a ____-____ linkage with the ε-amino group of a specific Lys residue at the enzyme’s active site

Schiff-base

Transaminations proceed through _____ steps to form PMP

Three

Aspartate aminotransferase is an archetypal _______-dependent transaminase

pyridoxal

The mitochondrial enzyme aspartate aminotransferase is a dimer with identical subunits, each consisting of a large domain and a small one

dimer

• PLP binds to the ___ 258 of the large domain, in a pocket

near the domain interface, in a Schiff-base linkage.

• A conserved ___ residue in the active site orients the

substrate appropriately in the active site.

• ____ 258 appears to act as the proton donor and acceptor.

Lys; Arg; Lys

The active site of aspartate aminotransferase reveals PLP bound to a lysine through a Schiff-base linkage

PLP

What is the role of pyridoxal

phosphate (PLP) in amino acid degradation?

a. PLP catalyzes the transfer of an α-amino group to an α- ketoacid.

b. PLP catalyzes the transfer of the amino group of alanine to α-ketoglutarate.

c. PLP allosterically stimulates glutamate dehydrogenase.

d. PLP forms Schiff-base intermediates in aminotransferases.

e. PLP orients substrates in the active site by binding to their α-carboxylate groups.

c

The presence of alanine and aspartate aminotransferase in the blood is an indication of ____ _____

liver damage

___ _____ can occur due to:

– viral hepatitis.

– long-term excessive alcohol consumption.

– reaction to drugs (e.g., acetaminophen).

Liver damage

In cases of liver damage, liver cell membranes are damaged, and ______ leak into the blood

aminotransferases

At the __-______ of amino acids, PLP-dependent enzymes catalyze:

– decarboxylations.

– deaminations.

– racemizations.

– aldol cleavages.

α-carbon

At the __-_____ and __-______of amino acids, PLP-dependent enzymes catalyze elimination and replacement reaction

β-carbon; γ-carbon

A Schiff base is formed by the ____ group (the amine component) and ____ (the carbonyl component)

amino; PLP

Protonated PLP acts as an electron sink to stabilize ______ intermediates that are negatively charged

catalytic

The product _____ base is cleaved at the completion of the reaction

Schiff

PLP enzymes labilize one of the _____ bonds at the α-carbon atom of an amino acid substrate

three

_______ effects determine specific bond cleavage

Stereoelectronic

Stereoelectronic control

the means of choosing one of several catalytic results

Bond ______ in the active site determines bond cleavage in different PLP enzymes

orientation

Serine and threonine can be directly _______

deaminated

____ dehydratase and ______ dehydratase directly deaminate their respective amino acids and PLP is the prosthetic group

Serine; threonine

_______ precedes deamination

dehydration

Peripheral tissues transport ______ to the liver

nitrogen

Muscle uses _______-chain amino acids as fuel during prolonged exercise and fasting

branched