enzymology part 2. principles of enzymology transition state theory: colliding molecules of the...
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PRINCIPLES OF ENZYMOLOGY
TRANSITION STATE THEORY:Colliding molecules of the reactants must have sufficient energy to overcomea potential energy barrier (the activation energy) to react
Factors affecting rate of a rxn:a. Sustrate concentrationb. Temperaturec. pHd. Inihibitors, effectors
PART A ([S] is low while [E] is high)
• Directly proportional• Initial velocity (
0) increases as [S] is small
compared to [E])• If [E] is higher than [S] in this section of the
graph, the frequency of collision is high
A. SUBSTRATE CONCENTRATION
AB
PART B ([S] is high while [E] is low
• Rate no longer depends on [S] • [S] higher than [E] • Active site is saturated with substrate• Frequency of collision no longer the
determining factor
Determination of 0
All the test tubes have the same contents except that each has a different substrate concentration
Either monitor the disappearance of substrate or the formation of product.Therefore can obtain the rate of disappearance of S or formation of P at each substrate concentration
k1 k3
E + S ES E + P Km = (k2+k3)/k1
k2 k4
• Km = substrate concentration at ½Vmax
• Units of Km = dm-3
• Vmax [S]
o = -------------- Km + [S]
0 = initial velocity
Vmax = maximum velocity (achieved when all the substrate molecules complex with E or enzymes are saturated with substrates)
• Km & Vmax are constants which are unique for a pair of enzyme and its substrate
Km = Michaelis constant (takes into account all the rxn constants of the rxns involved
MICHAELIS MENTEN EQUATION
• Km can be determined graphically
• Km shows the affinity of an enzyme for a substrate
• Km & Ks inversely related
• The bigger the value of Km, the lower the affinity
• Small Km value, the higher the affinity
LINEWEAVER-BURK PLOT
Km and Vmax can be determined from the vs [S]
The Michaelis Menten equation can be modified to obtain a more Km and Vmax precise values
1/0 = [Km/Vmax]. 1/[S] + 1/Vmax
ENZYME ACTION CAN BE CONTROLLED BY:Non covalent inhibition:2. Competitive inhibition3. Non competitive inhibition4. Uncompetitive inhibit5. Covalent inhibition: Irreversible6. Allosteric Control
COMPETITIVE INHIBITION Inhibitor competes with the substrate for the active site of the same
enzyme
Inhibitor and substrate have similar chemical structures
Lack of specificity at the active site: Cannot differentiate inhibitor from substrate
Inhibitory effect can be overcome by increasing substrate concentration
Note that:Vmax (- I) denoted V’max (in the presence of Competitve I)Km < K’m Because I and S compete for the same site. More S is required to achieve half saturation point. Hence Km increases. K’m is more than Km by a factor of (I + [I]/Ki)V’maks = Vmaks (I + [I]/Ki)
When enzyme is saturated with I, the effect can be overcome by increasing [S]. So when the enzyme becomes saturated with the S then Vmax is achieved.
SulphanamidePABA (para amino benzoic acid
Example: inhibition of folic acid synthesis by sulphanamide
PABA is required for the synthesis of folic acid Sulphanamida is a drug that competes with PABA for the enzymes in the folic acid synthesis pathway.
Find another example of competitive inhibition in the cell
Non-Competitive Inhibition1. The inhibitory effects cannot be overcome by increase in Substrate
concentration
2. Inhibitor binds to a site other than the active site. Binding of I does not affect binding of S
3. Therefore in this case, structure of inhibitor is not similar to substrate
4. Inhibitory effects depend on I and Ki and not on [S]
5. I can bind to either E or ES
I
EI
Gradient = Km/Vmax I/V’max = (I + [I]/KI)(I/Vmax
Vmax reduces in the presence of non competitive inhibitor. It is as though the amount of E is now less
Km does not change
Uncompetitive Inhibition
1. Binds only to the ES complex but not the free enzyme
2. Increasing [S] will increase the [ES] thus increasing [S] will not reverse the effects of an uncompetitive inhibitor.
3. Lineweaver Burke plots will give a set of parallel lines
Note: Both intercepts change but slope remains constant
Irreversible Inhibition
1. Enzymes can be inhibited by an irreversible manner for example by covalent attachment either to E or ES
2. Kinetic pattern looks like non-competitive inhibition (net effect is a loss of active enzyme): Vmax decreases
3. Reaction is time dependent decrease in enzymatic activity ie not instantaneous as seen in non competitive inhibition
4. Penicillin is an irreversible inhibitor: binds to serine residue in the active site of a the enzyme (glycoprotein peptidase). Affects cell wall synthesis, making bacterial cells susceptible to rupture.
5. Others include: Hg2+, Pb2+, arsenic
6. Binds to functional groups such as: –COOH, -NH2, -SH dan -OH
Effect of Temperature: Temperature increases the rate of reaction
Reaction rates increase because of the increase in collision/min between the substrates
BUT at extreme temperatures, enzyme activity decreases because of enzyme denaturation
Effect of pH
• Influences ionisation of functional groups in proteins
• As pH changes, the ionisation state of the functional groups of the amino acids in the tertiary structure and in the active site will change affecting enzyme activity
ENZYME REGULATION
Enzyme activity can be regulated by:
1. Covalent modificatio: eg phosphorylation of protein kinase
2. Zymogens
3. Allosteric regulation: non covalent interaction between enzymes and small molecules (metabolites)
In allosteric regulation enzyme activities controlled at key steps in metabolic pathways:
Feedback inhibition (feedback regulation)
The enzyme involved is a regulatory enzyme
Characteristics of Allosteric Enzymes
1. They have 2 binding sites: the active site and the modulator site
2. High molecular weight
3. Very complex
4. Difficult to purify
5. Contains 2 or more polypeptide chains (subunits): more than 1 S- binding site/enzyme molecule
6. Does not obey Michaelis Menten kinetics: vs [S] yields a sigmoid graph compared to a hyperbolic curve
The binding of 1 substrate to a protein molecule makes it easier for additional substrate molecules to bind to the same protein: substrate binding is termed cooperative
Characteristics of Allosteric Enzymes (con’t…)
6. Allosteric enzymes are regulated by activation: ie there are effectors or modulators that can have a positive (stimulatory) or negative effects on enzyme activity
POSITIVE COOPERATIVE EFFECT
and
NEGATIVE COOPERATIVE EFFECT
These enzymes take some time to reach saturation point.
MODEL TO EXPLAIN ALLOSTERIC ENZYME KINETICS
1. SYMMETRY MODEL: Monod, Wyman and Changeux
a. Allosteric proteins exist in two conformational stages:
R = Relaxed (high affinity for substrate) and T = Taut (low affinity for substrate
b. Model named symmetry because in each protein molecule all the subunits have either R or T conformation
c. These 2 states are in equilibrium
R0 T0
1. SYMMETRY MODEL: Monod, Wyman aand Changeux (con’t…)
d. Presence of S will result in binding to R0 to form R1. This reduces the R0 concentration of disturbing theT0/R0 equilibrium. To restore equilibrium, molecules in the T0 conformation will change to the conformation R0
e. Positive homotropic effectors
f. This model also provides for binding to positive and negative effectors. If effctors are not substrates then they are known as heterotropic effectors
g. Effectors that promote Substrate binding are known as positive heterotropic effectorsh. Effectors that diminish Substrate binding are known as negative
heterotropic effectors
i. Positive effectors increase number of available binding sites
j. Negative effectors decrease number of available binding sites
2. SEQUENTIAL MODEL: Koshland, Nemethy and Filmer
(Involves cooperativity and Conformational Changes)
Basis:1. Protein molecules are not in symmetry (ie asymmetric).
2. Proteins are flexible molecules and conformations altered when ligands bind
3. Binding of a ligand to one subunit of a multimeric protein, would cause conformational changes to occur, and then through contacts with the other subunits cause their conformation to change.
4. As a consequence other subunits will have either greater or lesser affinity for the ligand