UREA CYCLE AND ENZYMES

BBY 1208-lecture 7
AMINO ACID OXIDATION AND THE
PRODUCTION OF UREA

Oxidation

Waste or reuse
 AMMONIA HAS TO BE ELIMINATED
 Ammonia originates in the catabolism of amino acids
that are primarily produced by the degradation of
proteins – dietary as well as existing within the cell:
• digestive enzymes

• proteins released by digestion.

• muscle proteins

• Hemoglobin

• intracellular proteins
 AMMONIA HAS TO BE ELIMINATED
 Ammonia is toxic, especially for the CNS.

 Liver damage or metabolic disorders associated with

elevated ammonia can lead to tremor, slurred
speech, blurred vision, coma, and death

 Normal conc. of ammonia in blood: 30-60 µM

Nitrogen removal from amino acids

Transamination Aminotransferase
PLP

Oxidative
deamination

Urea cycle
 STEPS INVOLVED IN NITROGEN REMOVAL
FROM AMINO ACIDS
Step 1: Remove amino group

Step 2: Take amino group to liver for

nitrogen excretion

Step 3: Entry into mitochondria

Step 4: Prepare nitrogen to enter urea cycle

Step 5: Urea cycle

 EXCRETORY FORMS OF NITROGEN

a) Excess NH4+ is excreted as ammonia (microbes, aquatic

vertebrates or larvae of amphibia).
b) Urea (many terrestrial vertebrates).
c) or uric acid (birds and terrestrial reptiles).
 STEP 1. REMOVE AMINO GROUP
 Transfer of the amino group of an amino acid to an -keto
acid  the original AA is converted to the corresponding -
keto acid and vice versa:
 STEP 2: TAKE AMINO GROUP TO LIVER FOR
NITROGEN EXCRETION

Glutamate releases its amino

group as ammonia in the liver.

The amino groups from many of

Glutamate the -amino acids are collected in
dehydrogenase
the liver in the form of the amino
group of L-glutamate molecules.

The glutamate dehydrogenase of mammalian

liver has the unusual capacity to use either
NAD+ or NADP+ as cofactor
 NITROGEN CARRIERS
1. Glutamate
– transfers one amino group WITHIN cells:
– Aminotransferase → makes glutamate from -ketoglutarate
– Glutamate dehydrogenase → opposite
2. Glutamine
transfers two amino group BETWEEN cells → releases its
amino group in the liver
3. Alanine
transfers amino group from tissue (muscle) into the liver
 Move within cells
SynthAtase = ATP

Move between cells

In liver
 GLUCOSE-ALANINE CYCLE

Alanine plays a special role in transporting

amino groups to liver.

Ala is the carrier of ammonia and of the

carbon skeleton of pyruvate from muscle to
liver.

The ammonia is excreted and the pyruvate

is used to produce glucose, which is returned
to the muscle.

According to D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition

 SOURCES OF AMMONIA FOR THE UREA CYCLE

 Oxidative deamination of Glu, accumulated in the liver by the action of

transaminases and glutaminase

 Glutaminase reaction releases NH3 that enters the urea cycle in the liver (in the
kidney, it is excreted into the urine)

 Catabolism of Ser, Thr, and His also releases ammonia:

Serine →→ pyruvate + NH4+

Threonine →→ -ketobutyrate + NH4+

 Bacteria in the gut also produce ammonia.

STEP 3: ENTRY OF NITROGEN TO MITOCHONDRIA
STEP 4: PREPARE NITROGEN TO ENTER UREA CYCLE

Regulation
STEP 5: UREA CYCLE aspartate

Ornithine
transcarbamoylase

Argininosuccinate
synthase

Arginase 1

Argininosuccinate
lyase
 UREA CYCLE – SEQUENCE OF REACTIONS
 Carbamoyl phosphate formation in mitochondria is a prerequisite
for the urea cycle- Carbamoyl phosphate synthetase.

 Citrulline formation from carbamoyl phosphate and ornithine

 Ornithine transcarbamoylase.

 Aspartate provides the additional nitrogen to form

argininosuccinate in cytosol-Argininosuccinate synthase.

 Arginine and fumarate formation- Argininosuccinate lyase.

 Hydrolysis of arginine to urea and ornithine-Arginase.

DEFICIENCIES OF UREA CYCLE
ENZYMES
 AMMONIA ENCEPHALOPATHY

 Increased concentration of ammonia in the blood and other biological

fluids
 ammonia diffuses into cells, across blood/brain barrier

 Increased synthesis of glutamate from -ketoglutarate, increased

synthesis of glutamine

 -ketoglutarate is depleted from CNS → inhibition of TCA cycle and

production of ATP

 Neurotransmitters – glutamate (excitatory neurotransmiter) and GABA

(inhibitory neurotransmiter), may contribute to the CNS effects – bizarre
behaviour
 N-ACETYLGLUTAMATE SYNTHASE DEFICIENCY
 Deficiency or genetic mutation of enzyme (autosomal recessive)
 Associated with urea cycle failure.
 A severe neonatal disorder with fatal consequences, if not detected
immediately upon birth.
 Hyperammonemia and general hyperaminoacidemia in a newborn (liver
contain no detectable ability to synthesize N-acetylglutamate).
 Early symptoms include lethargy, vomiting, and deep coma.

Treatment with structural analog N-carbamoyl-L-glutamate – activates CPS-I,

mitigates the intensity of the disorder.
 CARBAMOYL PHOSPHATE SYNTHETASE (CPS I) DEFICIENCY
 Autosomal recessive metabolic disorder, associated with mental retardation
and developmental delay.
 Hyperammonemia has been observed in 0 – 50% of normal level of CPS-I
synthesis in the liver.
Treatment
 with benzoate and phenylacetate
 hippurate and Phe-Ac-Gln are excreted in the urine
 ARGININOSUCCINATE SYNTHASE DEFICIENCY –
CITRULLINEMIA (CITRULLINURIA)

 Autosomal recessive metabolic disorder.

 Caused by inability to condense citrulline with
aspartate.
 Accumulation of citrulline in blood and excretion
in the urine.
• Type I citrullinemia - usually becomes evident in
the first few days of life.
• Type II citrullinemia - the signs and symptoms
usually appear during adulthood and mainly affect
the nervous system.
Therapy – specific supplementation with arginine
for protein synthesis and for formation of
creatinine and ornithine.
 ORNITHINE TRANSCARBAMOYLASE (OTC) DEFICIENCY
 The most common urea cycle disorder, resulting in a mutated
and ineffective form of the enzyme.

 X-linked recessive disorder caused by a number of different

mutations in the OTC gene .
 Males are generally more seriously affected than females
(males are asymptomatic as heterozygotes).

 Complications with OTC may include mental retardation and

developmental delay.
 ARGININOSUCCINATE LYASE DEFICIENCY (ARGININOSUCCINATE
ACIDURIA)
 Rare autosomal recessive disorder.
 Argininosuccinate is excreted in large amount in urine.
 The severity of symptoms varies greatly, it is hard to evaluate the effect of therapy
– useful is dietary restriction of nitrogen.

ARGINASE DEFICIENCY (ARGININEMIA)

 Rare autosomal recessive disorder that cause many abnormalities in development
and function of CNS.
 Accumulation and excretion of arginine in urine and arginine precursors and
products of arginine metabolism.
Therapy – low nitrogen compounds diet (including essential amino acids
 ENZYMES
Proteins with catalytic properties due to
their power of specific activation
 FUNCTION OF ENZYMES
 Enzymes speed up the rate of chemical reactions in the
body; both breaking down (e.g.: starch into maltose)
and building up reactions. (e.g: amino acids into proteins)
 Enzymes lower the activation energy required to start a
chemical reaction
 Most enzymes are proteins.

 Enzymes are catalysts in living things.

 Enzymes are needed for almost all processes.

 ENZYMES AS CATALYSTS.
 Catalysts are substances that speed up chemical reactions.
◦ decrease the energy needed to start the reaction (activation
energy)
◦ increase reaction rate
 CHARACTERISTICS OF ENZYMES
 Enzymes are highly specific in action.

 Enzymes remain chemically unchanged at the

end of the reaction.

 Enzymes are required in minute amounts.

Proteinase
Substrate Name + -ase
Carbohydrate
ase
 ENZYMES CLASSIFICATION
 Oxidoreductases

 Transferases

 Hydrolases

 Lyases

 Isomerases

 Ligases
 OXIDOREDUCTASES

catalyze the transfer of hydrogen or oxygen

atoms or electrons from one substrate to
another.

Also called oxidases, dehydrogenases, or

reductases.
 HYDROLASES
catalyze hydrolytic reactions.

Includes lipases, esterases, nitrilases,

peptidases/proteases.

These are of the general form:

A-X + H2O ↔ X-OH + HA

 Lyases
 Catalyze non-hydrolytic removal of functional
groups from substrates.
 Often creating a double bond in the product; or
the reverse reaction, ie, addition of function
groups across a double bond.
 A-B → A=B + X-Y
X Y
 Includes decarboxylases and aldolases in the
removal direction.
 Synthases in the addition direction.
 ENZYMES CLASSIFICATION
TRANSFERASES
 catalyze group transfer reactions.
 These are of the general form:
A-X + B ↔ BX + A
ISOMERASES
 catalyzes isomerization reactions, including racemizations and
cis-tran isomerizations.
LIGASES
 Catalyzes the synthesis of various (mostly C-X) bonds, coupled
with the breakdown of energy-containing substrates, usually
ATP.
 DEFINITIONS
Substrate- monomers that bind to the
active site of an enzyme

Active site- area on enzyme where

substrate binds

Product- what the enzyme and

substrate binding produces.
 DEFINITIONS
Cofactors: An additional non-protein molecule that is
needed by some enzymes to help the reaction
 Tightly bound cofactors are called prosthetic groups
 Cofactors that are bound and released easily are called
coenzymes
 Many vitamins are coenzymes

Nitrogenase enzyme with Fe, Mo and ADP cofactors

Jmol from a RCSB PDB file © 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS

IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

 BASIC ENZYME DIAGRAM
The substrates have
reacted and changed
into the product

Enzyme is unchanged

Active site
 An enzyme’s structure allows only certain reactants to
bind to the enzyme.
– substrates
substrates
– active site (reactants)

enzyme

Substrates bind to an
enzyme at certain places called
active sites.
 ENZYME HYPOTHESIS
• Lock and Key hypothesis

• Induced fit hypothesis

 The Lock and Key Hypothesis
• This explains enzyme specificity
• This explains the loss of activity when
enzymes denature
• Fit between the substrate and the active site
of the enzyme is exact.
• Like a key fits into a lock very precisely
 THE LOCK AND KEY HYPOTHESIS
 The key is analogous to the enzyme and the substrate
analogous to the lock.

 Temporary structure called the enzyme-substrate complex

formed

 Products have a different shape from the substrate

 Once formed, they are released from the active site leaving it
free to become attached to another substrate
 LOCK AND KEY MODEL
Two substrates

Enzyme

Active site of the enzyme

 Lock and Key Model
The substrates fit like a key in a lock

Enzyme

The active site is like a lock

 MODE OF ACTION

Substrate fits in the enzyme active site,just like a key fits

into a lock.

An enzyme-substrate complex is formed.

Chemical reactions occur at the active site and products

are formed.
  The lock-and-key model helps illustrate how enzymes
function.
– substrates brought together
– bonds in substrates are weakened

Substrates bind to an The enzyme brings The catalyzed reaction forms

enzyme at certain places substrates together and a product that is released
called active sites. weakens their bonds. from the enzyme.
 INDUCED FIT HYPOTHESIS
Some proteins can change their shape
(conformation)
When a substrate combines with an enzyme, it
induces a change in the enzyme’s conformation
The active site is then moulded into a precise
conformation
Making the chemical environment suitable for
the reaction
 THE INDUCED FIT HYPOTHESIS

Hexokinase (a) without (b) with glucose substrate

 This explains the enzymes that can react with a range

of substrates of similar types
 FACTORS AFFECTING ENZYMES
substrate concentration

pH

Temperature

inhibitors
 THE EFFECT OF TEMPERATURE

Denaturation
Enzyme activity

0 10 20 30 40 50
Temperature / °C
 EFFECT OF TEMPERATURE
 For most enzymes the optimum temperature is
about 30°C
 Many are a lot lower, cold water fish will die at 30°C
because their enzymes denature

 A few bacteria have enzymes that can withstand very

high temperatures up to 100°C

 Most enzymes however are fully denatured at 70°C

EFFECT OF TEMPERATURE ON ENZYME ACTIVITY

At 0°C

 Low temperatures .

 low Kinetic Energy of enzymes and substrates.

 No/Very few enzyme-substrate complexes are formed.

 Enzymes are inactivated.

 20°C (INCREASING TEMPERATURE)
Increasing the temperature will lead to the increase
in kinetic energy of enzyme and substrate molecules.

Enzyme and substrate molecules move with

increasing speed and collide more frequently with
each other.

This increases the rate of enzyme-substrate complex

formation and product formation.

Rate of reaction increases

 At 37°C
As the temperature continues to
increase, the rate of enzyme activity also
increases until the optimal temperature
is reached.

Optimal temperature is the temperature

at which the enzyme works best.

 Rate of product formation is highest!

 >60°C- BEYOND OPTIMAL
TEMPERATURES
At high temperatures weak bonds within the
enzyme molecule are broken
Enzyme loses its shape and its active site.
Loss of shape leads to a loss of function.
Enzyme is said to have denatured
Denaturation : change in 3D structure of an
enzyme or any other protein caused by heat
or chemicals such as acids or alkali, causing it
to lose its function.
 DENATURATION

Different enzymes denature at different temperatures. Most enzymes denature at

temperatures higher than 60°C. However, there are some enzymes that stay active even at high
temperatures like 80°C (Enzymes in the bacteria Thermus aquaticus)
 EFFECT OF PH ON ENZYME ACTIVITY

Enzyme works best within a narrow pH

range
Each enzyme works best at particular pH,
known as its optimum pH level.
At extreme pH levels, enzymes lose their
shape and function and become
denatured.
 THE EFFECT OF PH
Extreme pH levels will produce denaturation
The structure of the enzyme is changed
The active site is distorted and the substrate
molecules will no longer fit in it
At pH values slightly different from the enzyme’s
optimum value, small changes in the charges of the
enzyme and it’s substrate molecules will occur
This change in ionisation will affect the binding of
the substrate with the active site.
EFFECT OF PH ON ENZYME
ACTIVITY
 The effect of pH
Optimum pH values

Enzyme
activity Trypsin

Pepsin

1 3 5 7 9 11
pH
 EFFECT OF SUBSTRATE ON ENZYME ACTIVITY

Faster reaction but it reaches a saturation point when all the

enzyme molecules are occupied.

 An enzyme solution has a fixed number of active sites to

which substrate can bind.
 At high substrate concentrations, all these sites may be
occupied by substrates or the enzyme is saturated.
 Substrate concentration: Non-enzymic reactions

Reaction
velocity

Substrate concentration
• The increase in velocity is proportional to the
substrate concentration
Further reading-Enzyme Kinetics
Enzyme Kinetics:
Study the rate of enzyme catalyzed reactions.

- Models for enzyme kinetics

- Michaelis-Menten kinetics
- Inhibition kinetics
 INHIBITORS
Inhibitors are chemicals that reduce the rate
of enzymic reactions.
Usually specific and they work at low
concentrations.
They block the enzyme but they do not usually
destroy it.
Many drugs and poisons are inhibitors of
enzymes in the nervous system.
TYPES OF INHIBITION
 Competitive Inhibition

 Noncompetitive Inhibition

 Uncompetitive Inhibition

 Irreversible Inhibition
 .

COMPETITIVE INHIBITION
 The inhibitors compete with the substrate molecules for the
active site. Enzyme
S
I

 Inhibitor resembles the substrate’s structure closely.

 Inhibitor binds only to the free enzyme, not to the ES complex

E + S ES E + P
+
I

EI
PRACTICAL CASE: METHANOL POISONING

 A wealthy visitor is
taken to the emergency
room, where he is
diagnosed with methanol
poisoning.
 You are contacted by a
3rd year medical student
and asked what to do?
 How would you suggest
treating this patient?
 Methanol (CH3OH) is metabolized to
formaldehyde and formic acid by
alcohol dehydrogenase.
 Advise the third year student to get
the patient very drunk.
 Ethanol (CH3CH2OH) competes with
methanol for the same binding site on
alcohol dehydrogenase.
 It slows the metabolism of methanol,
allowing the toxic metabolites to be
disposed of before they build up to
dangerous levels.
 NONCOMPETITIVE INHIBITION
 Inhibitor binds enzyme irregardless of whether the
substrate is bound
The inhibitor does not interfere with substrate binding
(and vice versa)
E + S ES E + P
+ +
I I

EI + S
.

ESI
I I
S
Enzyme S Enzyme

S
I I
S
Enzyme Enzyme
 UNCOMPETITIVE INHIBITION
 The inhibitor binds only to the ES complex, it does not bind
to the free enzyme
E + S ES E + P
+
I

ESI
Enzyme .

Enzyme

S
S
Enzyme
I I

Enzyme

I S
 IRREVERSIBLE INHIBITION

 Inhibitor permanently inactivates the enzyme.

 Inhibitor binds to the enzyme irreversibly through
formation of a covalent bond with the enzyme ,
.

permanently inactivating the enzyme

E + S ES E + P
+
I
Enzyme

EI
S
O I
 Summary-Enzyme Inhibition
 Competitive Inhibitor
◦ Binds to substrate binding site
◦ Competes with substrate
 Noncompetitive inhibitor
◦ Does not compete with the substrate for binding to the
enzyme
 Uncompetitive Inhibitor
◦ Binds to the enzyme only after the substrate has bound
 Irreversible Inhibitor
◦ Covalently modifies and permanently inactivates the
enzyme