5. Protein Function and Enzymes Part II Flashcards
Enzyme Kinetics
Define the terms initial velocity (V0), maximum velocity (Vmax) and Michaelis constant (KM).
initial velocity (Vo) = Vo is the initial rate of a reaction (at a given [S])
Maximum Velocity (Vmax) = is the maximum reaction velocity when all the enzyme is saturated with substrate and additional substrate changes the rate of the reaction less and less
Michaelis Constant (KM) = is the [S] when Vo is 1/2V max
Enzyme Kinetics
- Define KM and Vmax in terms of rate constants (k) and enzyme concentration.
E + S ⇌(k1 / k-1) ES ⇌ (k2/k-2) E + P
Km = (k-1 + k2)/k1 (breakdown of ES/Formation of ES)
- Michaelis Constant reflects the balance between formation and breakdown of ES complex.
Vmax = k2[Etotal]
- Maximum velocity is determined by the total concentration of enzyme in the system.
Enzyme Kinetics
Explain the shape of the graph of V0 versus substrate concentration in terms of formation of an enzyme-substrate complex (ES).
The shape of Vo vs. [S] plot resembles a hyperbola, as [S] increases, the [ES] increases as well.
The point (Vmax) at which [S] causes less and less change in Vo, is when [ES] has reached its maximum. (enzymes are all saturated)
Km = [S] @ Vmax/2
Enzyme Kinetics
Describe the Michaelis-Menten equation and the assumptions in its derivation.
Vo = Vmax[S]/(Km+[S])
- Reflects the balance between formation and breakdown of ES complex
- One substrate,
- enzyme catalyzed reaction,
- quantitative relationship between Vo and Vmax and initial [S], all reacted through Km
Enzyme Kinetics
- Manipulate the Michaelis-Menten equation (eg. derive Lineweaver-Burk equation).
Vo = Vmax[S]/(Km + [S])
1/Vo = (Km + [S]) / (V max[S])
1/Vo = Km/(V max[S]) + [S]/V max[S]
1/Vo = Km/ (V max[S]) + 1/(Vmax)
Enzyme Kinetics
Determine KM and Vmax from a Lineweaver-Burk plot.
Lineweaver-Burk equation is useful for a double-reciprocal plot of enzyme reaction rates, 1/Vo = Km/Vmax[S] + 1/Vmax Slope of this linear relationship is = K m/V max. Y-Intercept = 1/V max on 1/V o axis X-Intercept = 1/Km on 1/[S] axis.
Enzyme Kinetics
- Define the terms turnover number (kcat) and specificity constant (kcat/KM) and calculate them from kinetic data.
Turnover Number (kcat) - the ability of an enzyme to catalyze product when enzyme is fully saturated with substrate.
- Normalized value of Vmax
kcat = V max/[E total]
↑Kcat → ↑Specificity constant → better enzyme
specificity constant = kcat/Km
- Reflection of efficiency of BOTH binding and conversion
Specificity constant Kcat/Km can idicate “catalytic perfection”
Enzyme Kinetics
Describe kcat and KM and how they may be complex combinations of multiple rate constants, depending on the enzyme mechanism being studied.
kcat and Km vary among different enzymes because Km depends on rate constants and each enzyme may have a different mechanism or series of reactions.
Enzyme Inhibition
Define the terms α and KI, α’ and KI’.
α:
- Reflects how much substrate concentration must be changed to overcome inhibition
- the factor by which Km increases in the presence of a competitive inhibitor
- Vmax is not affected
- diagnostic of a competitive inhibitor:
α = 1 + [Inhibitor]/KI
KI:
- competitive inhibitor binding equilibrium constant
- Inhibitor dissociation constant (Kd for inhibitor)
KI = [E][I]/[EI]
α’:
- the factor by which Vmax is affected by a uncompetitive inhibitor.
- α’ = 1 + [I]/K’I
K’I:
- uncompetitive inhibitor binding equilibrium constant
- K’I = [ES][I]/[ESI]
Uncompetitive: Inhibitor binds site distint from substrate binding site and only binds ES complex
Competitive Inhibitor: Inhibitor resembles substrate and competes with it for binding to enzyme active site (binds E)
Enzyme Inhibition
- Write the equations relating the values of α to KI and α’ to KI’.
α = 1 + [Inhibitor]/KI
KI = [E][I]/[EI]
α’ = 1 + [I]/K’I
K’I = [ES][I]/[ESI]
α:
- Reflects how much substrate concentration must be changed to overcome inhibition
- the factor by which Km increases in the presence of a competitive inhibitor
- Vmax is not affected
- diagnostic of a competitive inhibitor:
α = 1 + [Inhibitor]/KI
KI:
- competitive inhibitor binding equilibrium constant
- Inhibitor dissociation constant (Kd for inhibitor)
KI = [E][I]/[EI]
α’:
- the factor by which Vmax is affected by a uncompetitive inhibitor.
- α’ = 1 + [I]/K’I
K’I:
- uncompetitive inhibitor binding equilibrium constant
- K’I = [ES][I]/[ESI]
Enzyme Inhibition
- Describe the differences between competitive, non-competitive and mixed inhibition.
Competitive Inhibitor - competitive inhibitor binds enzyme at the active site, to compete with the substrate
- Increases Apparent Km
- Michaelis-Menton Equation: Vo = Vmax[S]/(αKm + [S])
- Apparent Vmax = Vmax
- Apparent Km = αKm
Uncompetitive Inhibitor - inhibitor binds at a site other than the active site.
- Binds only to the ES complex.
- Decreases Apparent Km and Vmax
- Michaelis-Menton Equation: Vo = Vmax[S]/Km + α’[S]
- Apparent Vmax = Vmax/α’
- Apparent Km = Km/α’
Mixed Inhibitors
- binds at sites distinct from substrate active sites, but it binds to either E or ES.
- Michaelis-Menton Equation: Vo = Vmax[S]/αKm + α’[S]
- Apparent Vmax = V max/α’
- Apparent Km = αK m/α’
α = 1 => No inhibitor [I]=0
Enzyme Inhibition
Understand the difference between reversible and irreversible inhibitors and their effects on the Michaelis-Menten equation, the KM and Vmax values.
Reversible Inhibitors bind reversibly to the enzyme
Irreversible inhibitors bind covalently with the enzyme and destroy the functional group on an enzyme that is essential for the activity of an enzyme.
Enzyme Inhibition
Explain why transition state-analogs can be effective inhibitors.
Transition state analogs can be used as inhibitors in enzyme-catalyzed reactions by blocking the active site of the enzyme. Theory suggests that enzyme inhibitors which resembled the transition state structure would bind more tightly to the enzyme than the actual substrate.
Enzyme Inhibition
Use Lineweaver-Burk plots to distinguish kinetically among different types of inhibitors.
a > a’ = Competitive Inhibition
KI<KI’ = increase affinity for E over ES
a’>a = Uncompetitive
Increase affinity for ES
a=a’ = non-competitive
Control of Enzyme Activity
List mechanisms used by organisms to regulate enzymes.
Allostery (R/T)
- Feedback inhibition
Covalent modification
- Phosphorylation
- Adenylylation (Tyr)
- ADP-ribosylation
- Palmitorylation (lipid anchor)
Regulatory Enzymes - enzymes which have increased or decreased catalytic activity in response to certain signals. In multi enzyme systems the first enzyme of a sequence is typically a regulated pathway to prevent unneeded products from forming unless the pathway is active.
Control of Enzyme Activity
Outline general features of allosteric enzymes.
- Enzymes do not obey Michaelis-Menten kinetics
- Sigmoidal activity curves are typical
- Cooperative substrate binding (positive homoallostery)
- Two (at least) states: high and low activities (R and T states)
- Often associated with feedback inhibition
Features:
* Allosteric proteins have quaternary structure.
* Each oligomer can exist in either R or T state (high and low activity respectively).
* Ligands/substrates will bind to both states (with different affinities) and binding shifts equilibrium between T and R states.
* The overall protein symmetry is maintained (i.e. All subunits are in the same conformation).
Control of Enzyme Activity
- Define the terms homotropic and heterotropic effector, positive and negative effector, feedback inhibition.
Homotropic Modulator - modulators of allosteric enzymes which are identical to the substrate.
Heterotropic Modulator - modulators of allosteric enzymes which differ from the substrate the enzyme acts upon.
Positive Modulators - modulators which are stimulatory. When bound to the enzyme results in increased binding of the substrate.
Negative Modulators - modulators which are inhibitory. When bound to the enzyme results in decreased binding of the substrate.
Control of Enzyme Activity
- Describe general kinetic differences between allosteric and non-allosteric enzymes.
Allosteric Enzymes have sigmoidal saturation curves, whereas with non-allosteric enzymes they experience hyperbolic saturation curves
Control of Enzyme Activity
Outline and describe features of the allosteric enzyme aspartate transcarbamoylase in terms of kinetics, effectors and structure.
Aspartate transcarbamoylase (ATCase)
* EC2.1.3.2 (2 = Transferase)
* Quaternary structure
* 6 catalytic subunits
* 6 regulatory subunits
* Symmetry: D3 (12 subunits; 6 protomers)
* Catalyzes First step in CTP biosynthesis
* Feedback inhibited (CTP) (Heterotropic Inhibitor)
* Activated by ATP (Heterotropic Activator)
* Generally follows the symmetry model
* Aspartate is homotropic activator
Control of Enzyme Activity
Explain the physical origin of the sigmoidal curve of [S] versus V0 for a homotropic allosteric effector.
Homotropic Allosteric Enzymes are generally multi-subunit proteins and the active site is also the regulatory site. Sigmoidal kinetics signifies that there is cooperative interactions between protein subunits. Changes in the structure of one subunit are translated into structural changes in adjacent subunits. Binding of substrate to one subunit enhances the next substrate to bind.
Control of Enzyme Activity
List some of the possible effects of a heterotropic effector on the sigmoidal curve, K0.5 and Vmax for an allosteric enzyme.
Activator
* can cause curve to become nearly hyperbolic,
* decrease in K0.5
* no change in Vmax,
* resulting in an increased reaction velocity at a fixed [S].
* can cause an increase in Vmax with little change in K0.5
Inhibitor
can produce a more sigmoidal substrate-saturation curve with an increase in K0.5.
Control of Enzyme Activity
Outline and describe features of the allosteric enzyme phosphofructokinase in terms of kinetics, effectors, structure and mechanism.
ATP:
- Binds at active site and regulatory site
- Homotropic inhibitor = decreases activity
Fructose-6-phosphate
- Binds to active site
- Homotropic Activator
- Sigmoidal Relationship
AMP:
- Activator (Heterotropic)
- Same regulatory site as ATP
Phosphoenolpyruvate
- Heterotropic Inhibitor
homotropic allosteric effector is a substrate for the enzyme, as well as a regulatory molecule
Control of Enzyme Activity
Differentiate between reversible and irreversible covalent modification.
Reversible Covalent Modification - covalent modification can be added and removed,
irreversible covalent modification, group cannot be removed. e.g. Protelytic cleavage of Chymotrypsinogen
Control of Enzyme Activity
- Outline and draw simple reactions for two reversible covalent modifications that use ATP as a substrate.
Regulation of Glycogen Phosphorylase by phosphorylation
- Glycogen phosphorylase is activated by phosphorylation by specific kinase
- Enzyme + ATP → Enzyme-P + ADP
Adenylation (Tyr)
- Enzyme + ATP → Enzyme-AMP + PPi (pyrophosphate)
Glycogen Synthase is regulated by multiple regulatory phosphorylations. This allows for extremely subtle modulation of enzyme activity. It is modulated by at least 5 protein kinases.
Control of Enzyme Activity
Describe the physical interactions that may lead to conformational change when a phosphate group is added to a protein structure.
Active site can undergo changes in structure and change catalytic activity when phosphorylated vs. non-phosphorylated form. Phosphorylation can also affect the enzyme’s catalysis by affecting the substrate-binding affinity.
Control of Enzyme Activity
Define the term “target sequence” in the context of protein kinases.
In the context of protein kinases, the term “target sequence” refers to a specific amino acid sequence in a protein substrate that is recognized and phosphorylated by the protein kinase. Protein kinases are enzymes that catalyze the transfer of phosphate groups from ATP molecules to specific amino acids (usually serine, threonine, or tyrosine) in target proteins.
Some kinases need to recognize neighbouring amino acid residues (consensus sequences) e.g. Proline
Amino acid sequence and 3-D structure giving availability to amino acid residues to be phosphorylated.
Hierarchical phosphorylation: certain residues are only phosphorylated when other residues are phosphorylated first.
Control of Enzyme Activity
Describe the process of zymogen or proenzyme activation using chymotrypsin as an example.
Zymogens (unaltered enzymes) are activated by modification by other enzymes
* Digestive proteinases are modified by other proteinases
* Enzymes must then be destroyed or inactivated
Zymogens = inactive enzyme precursor
- require specific proteolytic cleavage to become active. chymotrypsinogen, the inactive precursor of chymotrypsin .
- Cleavage: Trypsin cleaves a small peptide fragment from the N-terminal of chymotrypsinogen, exposing an active site for subsequent cleavage.
Formation of a functional active site occurs once the peptide is cleaved after residue 15 (oxyanion hole forms).
a-chymotrypsin is the fully mature form
Autocatalysis: The newly exposed active site of chymotrypsinogen undergoes autocatalytic cleavage, resulting in the removal of a larger peptide fragment and the formation of the active enzyme chymotrypsin
Feedback inhibition: Chymotrypsin in turn can act on other chymotrypsinogen molecules to produce more active enzyme. However, once enough chymotrypsin has been produced, it can also inhibit the further activation of chymotrypsinogen, thus regulating its own activity.
