Chapter 6: Introduction to Enzymes Flashcards
Enzyme
a biomolecule, often a protein but sometimes RNA, that catalyzes a specific chemical reaction
Substrate
a specific component acted on by an enzyme(reactant)
Cofactor
an additional chemical component that binds to the protein and aids in substrate binding and/or catalysis
- may be metal ions or coenzymes
Enzyme with cofactor(s)
Holoenzyme
- active
enzyme without cofactor(s)
Apoenzyme
- inactive
Active site
the pocket on the enzyme where the substrate binds and catalysis happens
- cleft or hollow
- water is often excluded
- small part of protein structure
Coenzyme
an organic cofactor, often derived from vitamins
ex: heme, ubiquinone, FAD
-subtype of cofactor
Prosthetic Group
coenzyme or metal ion that is very tightly or covalently bound to the enzyme protein
- permanently bound
How do enzymes work?
accelerate a chemical reactions but do not change the equilibrium
- reduce the activation energy
transition state
: fleeting molecular moment, often mid bond break or form
What does the enzyme rate depend on?
- concentration of substrates
- rate constant
First Order Rate Equation
V=k[S]
k units=s-1
Second Order Rate Equation
V=k[S1][S2]
k units=s-1M-1
Substrate Specificity
ability to discriminate between substrate and related compounds
Enzymes that lack specificity
called promiscuous
Enzyme Catalysis Strategies
- desolation from the substrate
- orientation and approximation (stabilizing transition state)
- (Temporary) covalent interactions between the enzyme and the substrate
-Metal-ion assisted - General Acid-base catalysis
Stabilizing the Transition State
- the enzyme is complementary to the transition state, not the substrate
- favoring/ pushing substrate towards desired transition state and product - binding is often favorable, free energy of binding can drive reaction
General Acid-Base Catalysis
- H+ transfer
- A H+ transfer can be used to stabilize a charged intermediate or transition state
What is enzyme kinetics the study of?
Enzyme kinetics is the study of the rate of an enzyme-catalyzed reaction under various conditions
-provides quantitative information
What is modern enzyme-kinetics based on?
steady-state condition i.e. when the [ES] is constant
Enzyme kinetics graph axis’s
- product concentration vs time
- y-axis, x-axis
Why do we only use initial velocity?
We use initial velocity because the concentration of the substrate hasn’t changed very much and the situation gets more complicated as we decrease [S] and increase [P]
What happens to velocity with increasing [S]?
velocity increases to a maximum (Vmax)
Michaelis Menton Equation
an equation to describe the kinetic plot of the rectangular hyperbola produced by plotting Vo vs [S]
Vo=(Vmax[s]/Km+[S])
Enzymes that display a rectangular hyperbola graph are said to have __________.
Michaelis-Menten Kinetics
Vmax
the maximum velocity at near saturating [S] (all E exists as ES)
Km
- Michaelis Constant
- [S] at 1/2 Vmax
- combination of rate constants
Km=(K2+K-1)/(K1)
What two assumptions are made in Michaelis Menten Kinetics?
- Steady State Assumption: the concentration of [ES] is constant
- Rapid Equilibrium: the formation of the ES complex is fast and product formation is slow (rate limiting)
When can Km be equal to Kd (binding affinity of E+S)?
If k2«««k-1
- this means ES->E+P is much slower than E+S->ES
What is Kd?
Dissociation constant= affinity/binding
kcat
the rate constant for the rate limiting step at saturating [S]
- assuming Michaelis Menten kinetics, k2=kcat
turnover number (aka kcat)
the number of substrate molecules catalyzed per second
- units of s-1 (first order)
Lineweaver-Burk Plots
- also called double-reciprocal plots
- plots the reciprocal of Vo and [S]
How do we look at enzyme kinetics if the enzyme has two substrates?
We place one substrate in great excess over the other substrate so that the rate only depends on one of the substrates.
Km(apparent)
- The Km measured in the presence of an inhibitor
= alpha x Km
How do competitive inhibitors work?
they bind to the active site and compete with the substrate
- increase Km
- no effect on Vmax because as high [S], natural substrate can outcompete inhibitor
How do uncompetitive inhibitors work?
they do not bind at the active site, they only bind to the ES complex, not E alone
- decrease the Km
- decrease the Vmax
How does mixed inhibition work?
Mixed inhibitors do not bind at the active site and can bin either the E or the ES complex
- May increase or decrease the Km
- Decrease Vmax
Irreversible Enzyme Inhibition
Enzymes can be irreversibly inhibited by covalent modification that destroys critical function groups OR by binding a molecule so tightly, it is essentially irreversible
Suicide Substrate/ Inactivators
are modified by the enzyme and then become covalently bound to the active site
Transition State Analogs
molecules that resemble the substrate transition state and bind with his affinity
Regulatory Enzymes
enzymes at key steps in a metabolic pathway
- heavily regulated
- exists an an equilibrium between an active and inactive conformation
How can enzyme activity be controlled?
- substrate availability
- allosteric modulators/effectors
- reversible covalent modification
- post translational modification (PTM)
- Proteolytic cleavage
Allosteric Effectors/ Modulators
generally small molecules that bind away from the active site and change the enzymes activity (can have a negative or positive effect)
- with Hb, 2,3-BPG is a negative allosteric modulator (favors T state)
Kinetics of allosteric enzymes?
- will not display Michaelis Menten kinetics, but instead will have sigmoidal plot of Vo vs [S]
What are Post-Translational Modifications?
- include the addition of various function groups to the side chains or termini of proteins
How do post translational modifications affect enzymes?
- can alter intermolecular forces, changing the enzyme structure and activity
- can activate or inhibit
Kinases
the enzymes that transfer a phosphate from ATP to a target protein (Ser, Thr, or Tyr residues)
Protein Phosphatases
cleave and remove phosphate groups
What does chymotrypsin do?
It is a protease, cleaves peptide binds adjacent to large aromatic side chains: Phe, Trp, Tyr
What are chymotrypsins substrate?
peptide and water
What class of enzyme does chymotrypsin belong to?
hydrolase
What is the structure of Chymotrypsin?
monomer (multiple chains), soluble and globular
- initially translated from RNA as one continuous polypeptide chain
Zymogen
inactive precursor
Chymotrypsinogen
inactive/not cut form of chymotrypsin
What happens when chymotrypsin is delivered to the digestive tract?
When delivered to the digestive tract, it is cleaved into three chains that are held together by disulfide bonds
What role does Ser195 play in the reaction mechanism of chymotrypsin?
-contains a strong nucleophilic O (alkoxide, stabilize His57) that attacks carbonyl carbon of substrate
-forms the covalent bond with substrate, facilitating peptide bond cleavage
What role does His 57 play in the mechanism
-multiple general acid-base mechanism
- protonated and deprotonated multiple times
- stabilizing the alkoxide of Ser195, also generating hydroxide (-OH) from H2O
What role does Asp102 play in the reaction mechanism
positioning His 57 in the right location and the negative charge helps stabilize the protonations of the His side chain
What common strategies does chymotrypsin use to catalyze the peptide cleavage?
General-acid base, covalent catalysis, approximation and orientation
What is the catalytic triad?
- Ser195, His 57, Asp102
