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Protein Part 5 Enzyme-2023-2024
Protein Part 5 Enzyme-2023-2024
Enzymes 25.11.2023
Denaturation / dissociation into subunits/ hydrolysis into a.a → lost activity → activity
dependent on structure ( primary, secondary, tertiary and quaternary.
- Mwt = 12,000 to million
- Measurement of enzyme activities in blood plasma and tissue samples→ diagnosing
example: liver enzymes
Some enzymes require no chemical groups for activity other than
their amino acid residues. (simple enzyme)
cofactors
Coenzymes
حامل مؤقت
sources of coenzymes
in vitamins supplied by food
coenzymes
= 2 carbons
FAD
NADH
NAD
PLP
Coenzymes act as transient carriers of specific functional groups.
Most are derived from vitamins, organic nutrients required in small amounts in the
diet.
thiol group
pyruvate to acetylCoA
any of a class of yellow water-soluble nitrogenous pigments derived from
isoalloxazine and occurring in the form of nucleotides as coenzymes
of flavoproteins; especially :RIBOFLAVIN
2e+2p
any of a class of yellow water-soluble nitrogenous pigments derived from
isoalloxazine and occurring in the form of nucleotides as coenzymes
of flavoproteins; especially :RIBOFLAVIN
hydride ion = 2e + 1 p
Enzyme names:
1) Addition of suffix “ ase” to a name describing substrate / function
Urease → urea hydrolysis
DNA polymerase → polymerization of DNA
Solution: System for naming and classification :6 classes, subclasses based on rxn catalyzed.
hydroxyl group on gluc
ATP + glucose → ADP + glucose -6 p kinase --> phosphorelation
(ATP:glucose phosphotransferase)
Other name hexokinase works on hexose sugar glucose
6
binds
Enzyme Classification
EE E E E
conformational change
E+S ES EP E+P
complex 1 complex 2
Biochemical systems
[H+] << 1M
P <<S
Biochemical standard -ve overall standard
Free-energy change
free energy change starting point In S→P directions.
∆G`° at pH 7
rxn progress (e.g., bond breakage or formation)
Any reaction may have several steps, involving the formation and
decay of transient chemical species called reaction intermediates.
intermediate converts quickly
- Enzymes are highly effective catalyst enhancing rxn rates by a factor of 105 -1017
breaks proteins
cyclophilin
CA not by heart
triose phosphate isomerase
carboxypeptidase
phosphoglucomutase
urase
orotidine
Rate is described by determining how quickly the reactant are consumed or the product is
formed.
s= substrate conc ➢ Rate equation
Rate / velocity V = k[S] , k= rate constant, unit : reciprocal time: s-1
reflects probability of rxn in a given temp, pH, etc…
Order of reaction describes how the rate of reaction depend on conc. of reactants
➢ The rate of any reaction is determined by the concentration of the reactant (or
reactants) and by a rate constant (constant not depend on concentration).
S → P , 1st order rxn
If a first-order reaction has a rate constant k of 0.04 (mean that 4% of the
available S will be converted to P in 1 s)
A reaction with a rate constant of 2,000 s1 will be over in a small fraction of a
second
V = k[S]
2) Induced fit :
Active site not 100% complementary to S but to transition states.
An imaginary enzyme (stickase) designed to catalyze breakage of a metal stick
ES complex is more
increase in AE
stable and has less free
rigid and not flexible and has to be breathing
energy in the ground
state than substrate
alone. The result is an
increase in the activation
energy.
No change in E or
substrate shape
more acceptable
enzyme must be
complementary to the
reaction transition state
More acceptable model.
Involve parts of the stick far away from bent site stabilize + Changes in shape
away from water. Explain why E are large?
Weak Interactions between Enzyme and
Substrate Are Optimized in the Transition State
Role of binding energy is catalysis:
activation energy for a rxn lowered by binding energy bw E and S weak
noncovalent interactions in transition state.
Acid- base catalysis: (general not specific –proton transfer involve molecules other than water)
In E active site several a.a serve as proton donors / acceptors (most common
catalysis type). Proton transfer provide rate enhancement of 102 – 105
glu asp
lys arg
cys
His
Ser
Tyr
Covalent catalysis:
Transient covalent bond formed bw S and E
A-B →(H2O)→ A+B (hydrolysis of the bond)
His 57
acts as a base
H+
electron-
deficient atom
(an
electrophile). Break peptide bond
OH in ser is not strong nucleophile so the H will be removed by His N atom, that
were oriented in the correct position by the attraction with Asp. i.e stabilize the
histidine so it can carry the protonation, and convert Ser to more nuleophile by
converting OH to alcoxide that attack the carbon of the carbonyl group . So break
down of peptide bond.
https://www.youtube.com/watch?v=OY1WsqlcUdo
Enzyme kinetics
Vo = Vmax [S]
Km +[S]
1 = [S]
2 KM + [S]
The equation
Vo = Vmax [S]
Km +[S]
Vo = 1/2 Vmax
Vmax = Vmax [S]
2 Km + [S]
Km= [S]
Equation and Km provide little information about chemical nature/ steps of the rxn.
Importance of Km in enzyme reactions
= s conc
catalase
hexokinase
CA
chymotrypsin
galactosidase
threonine dehdrase
Michaelis-Menten Equation
Lineweaver–Burk Plot
V0 = Vmax [S]
KM + [S]
slope
y intercept
Double reciprocal plot or Lineweaver-Burk plot: (more convenient)
The Michaelis-Menten equation
Vo = Vmax [S]
Km+[S]
1 = Km+[S]
Vo Vmax [S]
1 Important in analyzing
E inhibition.
2
Advantage: precisely
determining Vmax
Kcat = most of the enzyme is in the EP form at saturation, and Vmax = k3[Et]. describes the limiting
rate of E – catalyzed rxn at saturation. kcat = Vmax/[Et]. The concentration of enzyme is necessary.
Kcat = turnover number = tell us the maximum number of S molecules → P in a unit time on a single
active site of E molecule when E saturated with S
EX:A single active site converts 40,000,000 substrates to product per sec
Taking the reciprocal of kcat(1/kcat: 1/40.000,000) give the time that is needed for
one substrate to convert to product.
1/kcat = time in sec
Km: describes the amount of S needed for the E to obtain half of its
maximum rate of reaction.
2) kcat
The second step of catalysis kinetic is the forming of P.
• The larger kcat is, the more favorable the reaction towards P
Kcat and Km for evaluation of kinetic efficiency of enzymes
•HIGH (kcat is much larger than KM) and the enzyme complex converts a
greater proportion of the substrate it binds into product. (substrate binds
more firmly (strong) to the enzyme, a consequence of relatively low KM ) or
a large turnover rate kcat.
•LOW (kcat is much smaller than KM ) and the complex converts a lesser
proportion of the substrate it binds into product
Enzymatic rxn with 2 or more S :
Enzyme inhibition:
inhibitors: agents that interfere with catalysis slowing / halting
enzymatic rxns.
1) Pharmaceutical agents:
e.g aspirin (acetylsalicylate) inhibits prostaglandins synthesis
(pain processes).
2) Discovery and define of metabolic pathway.
Solution:
Add more S
[S]>>[I]
◼ Most common
◼ Inhibitor competes with natural substrate for binding to
active site
◼ Inhibitor similar in structure to natural substrate and binds
active site of enzyme (reducing effective enzyme conc)
◼ Binds more strongly
◼ May or may not react
◼ If reacts, does so very slowly
◼ Gives info about active site through comparison of
structures
Reversible Inhibition (competitive)
double receprocal
Vmax +Inh
1/v
-Inh ternary
+inh
vo
-Inh
shift to the right
1/2 Vmax in low affinity binding
doesn't change memorize the curve
larger Km
1/Vmax
slope is changed
Use of competitive inhibitor in Medical therapy:
Case:
methanol poisoning usually from contaminated alcohol beverages
toxic
Methanol in liver → alcohol dehydrogenase (EC 1.1.1.1) → formaldehyde → formic acid→ metabolic
acidosis
Symptoms:
Vomiting, abdominal pain, photophobia, tissue damage
Ingestion of 10ml →blindness , 30ml → fatal (2 tablespoons deadly to a child).
Treatment:
Antidote مضاد سميto reverse the effect of the poison intravenous infusion of ethanol =
ethanol is a competitive inhibitor
competitive inhibitor to alcohol dehydrogenase. ethanol is less dangerous than methanol
The dehydrogenase enzymes facilitate the interconversion between alcohols and aldehydes or ketones with the
. of nicotinamide adenine dinucleotide (NAD+ to NADH)
reduction
CH3OH + NAD+ CHO + NADH +H+ ethanol
CH3CH2OH + NAD+ → CH3CHO + NADH + H+ methanol
Uncompetitive inhibitors:
Bind at a separate site, but bind only to the ES complex ( after S)
α Km ↓ and Vmax ↓
Cyanide combines
with the prosthetic
group of cytochromo
in mitochondria
oxidase and inhibits
the election
transport chain
ping pong
1. Binds only to ES complex but not free enzyme
ןKm Km [Substrate]
Mixed inhibitors:
Also bind to a site separate than the active site, but may bind to
either E or ES .
ternary complex
same v max
Km inc
Inhibitor binds E or ES
◼ Increasing [I]
Vmax 1/v
-Inh
Vmax 1/Vmax -Inh
(app)_
+inh (app)
vo
1/2 Vmax km the same
v max is decreased
1/2 Vmax 1/Vmax
(app)
-1/Km 1/[S]
Km Km [Substrate]
(app)
uncompetitive
non competitive
1/v
+Inh
-Inh
mixed
-1/Km 1/[S]
Effects of reversible inhibitors on Km and Vmax
Uncompetitive ES
Mixed ES, E
Noncompetitive E
Irreversible inhibition: dentist
Binds irreversibly to E active site by forming a covalent bond/ very stable non
covalent interaction → destroy a functional group.
diisopropylflourophosphte (DIFP):
irreversibly inhibits chymotrypsin
Ser195 is the key active site residue in chymotrypsin.
Suicide inactivators / mechanism-based inactivators:
Relatively unreactive until binding to E active site.
Perform first few chemical steps of rxn→ not transformed to normal product.
• Inactivator is converted to a very reactive compound that combines irreversibly with the enzyme
in the liver
Regulatory Enzymes:
- One / more E in a metabolic pathway have great effect on the rate of
the overall rxn.
- Activity of allosteric E ( increase /decrease) regulated by reversible
binding of a specific modulator to a regulatory site other than active site.
Effect of modulator = positive / negative.
Modulators maybe the S itself (Homotropic modulator) / other
metabolites.
Kinetic behavior of allosteric E reflects cooperative interactions among E
subunits ( similar to O2 – hemoglobin).
Regulatory Enzymes:
Allosteric E regulated by reversible noncovalent binding of modulators
/ effectors.
Other E regulated by reversible covalent modification.
- All are (Multisubunit proteins). all reg are multisubunits
cata
cata
reg
cata
Subunit interactions in an allosteric E, interactions with inhibitors and
activators:
When M dissociates.
Enzyme→ inactive/ less
active.
substrate
In most multienzyme systems, the first enzyme binds to catalytic
of the sequence is a regulatory enzyme
reg enzyme
Feedback inhibition =
- The regulatory E in a metabolic pathway is
a b c d = intermediates
inhibited by the end product of the pathway.
- Build up of end product slows entire pathway
less common
Covalent modifications (reversible) of regulatory enzymes by a functional group