BT-202 Netaji Subhas Institute of Technology, Dwarka, New Delhi. Dr. Amita Pandey Sept 5, 2011
Regulation of enzyme activity
Louis Pasteur (1850s): ferments, vitalism
Eduard Buchner (1897): fermentation is done by molecules
Frederick W. Kuhne: coined the name enzyme
James Sumner (1926): isolated urease and said enzymes are made up of proteins.
J.B.S. Haldane: weak interactions between enzyme and substrate are important for catalysis
Chemical reactions in cells require
Enzymes are proteins which perform
- 12kDa - 1,000kDa or more
- larger than their substrate
Metabolite acted upon is called the enzyme ’s substrate.
- some residues involved in binding substrate
-others catalyze reaction
for some reactions, the amino acids are not powerful enough for catalysis. Enzymes overcome this by incorporate additional factors.
- metal ions as cofactors Zn 2+ , Fe 2+ , Cu 2+
-coenzymes are organic cofactors
Coenzyme or metal ion cofactor bound to enzyme either tightly or covalently.
Cofactor and Coenzymes
Properties of Enzymes
Enzymes are excellent catalysts
- speeding up reactions 10 8 to 10 20 fold
Not used up during a reactions
Specificity for substrate
- absolute (eg., DNA polymerase)
- broad range (eg., synthesis of secondary metabolites)
Regulated- some enzymes can sense
Classification of Enzymes
named and classified according to the substrate acted upon and the reaction catalyzed.
trivial names- end in –ase . (eg., urease, hexokinase.
based on a formal systemic catalog (IUB) with six major classifications.
Reaction Rates and the Transition State
In order to react, the molecules involved
are distorted, strained or forced to have
an unlikely electronic arrangement.
That is the molecules must pass through a
high energy state.
This high energy state is called the transition state .
The energy required to achieve it is called
the activation energy for the reaction.
The higher the free energy change for the transition barrier, the slower the reaction rate.
Enzymes lower energy barrier by forcing the reacting molecules through a different transition state.
Eg., C 12 H 22 O 11 + 12O 2 12CO 2 + 11H 2 O Reaction Intermediates Transient chemical species formed during a rxn Rate limiting step When many steps are involved the overall rate of the Reaction is determined by the step which has the Highest activation-energy
Relationship between reaction equilibrium and free energy Equilibrium constant is directly related to free energy Large negative free energy favors reaction
Rate of a reaction Unimolecular or first order reaction; V=k[S] Units are s -1 Rate for second order reaction; V= k[S 1 ][S 2 ] for transition state k = k T / h e - Δ G/RT Lower activation energy means faster rxn rates
How enzymes do it? Binding energy ( Δ G B ) Energy derived from enzyme substrate interaction. -transient covalent bond in the active site -non-covalent interactions to form ES
Role of binding energy in catalysis k = k T / h e - Δ G/RT Δ G + must be lowered by 5.7kJ/mol to accelerate first order reaction.
Specificity is derived from the formation of many weak interactions between the enzyme and its specific substrate.
Desolvation of substrate
Enzyme undergoes conformation changes i.e. induced fit, postulated by Daniel Koshland (1958)
Modes of Enzymatic Enhancement of Rates
Involve transient covalent interactions with a substrate or group transfer to or from a substrate
General acid-base catalysis
-good proton donors & acceptors positioned just right.
metal ion catalysis
-electron donor or acceptor
At low concentrations of [S] V o increases almost linearly
At higher [S] concentration V o increases by smaller amounts in response to increase in [S]
Finally increase in V o is negligible as [S] increases
Steady state kinetics
rate stops increasing or plateaus because the complex ES becomes filled at high [S]
Pre-steady state and steady state
Introduced by Briggs and Haldane
Lineweaver-Burk equation Double reciprocal plot
Interpreting V max and K m Steady state kinetics is the means by which biochemists can compare and characterize The catalytic efficiencies of enzymes.
K cat it is the limiting rate of any enzyme catalyzed reaction. Eg. , K cat is first order rate constant has units s -1 . It is also called Turnover number ( the number of substrate molecules converted to product) .
Specificity constant compares the catalytic
efficiencies of different enzymes or the turnover
of different substrates by the same enzyme.
V o = k cat / K m [E t ][S]
-V 0 depends upon [Et] and [S]
-k cat /K m is a second order rate constant
-units M -1 s -1
Reactions with more than one substrate
In a simple enzyme catalyzed rxn., the sum of
which two chemical species is strictly constant?
[E] + [ES]
[E] + [P]
[S] + [P]
[ES] + [P]
2. For a simple enzymatic rxn., what is the value of
The initial velocity when S=5km?
Match the following
Michaelis complex The velocity at t=0
Michaelis constant Km Also known as turnover number
Initial velocity The enzyme substrate complex
Maximal velocity The velocity when the enzyme is saturated with substrate
Catalytic velocity The conc. of substrate @ which the velocity is half maximal
rxns. are enzyme reactions with two substrates.
In Lineweaver-Burk plot of a simple enzymatic rxn. , what is the value of the y-intercept @ 1/Vo axis?
Regulatory enzymes Exhibit increased or decreased catalytic activity in response to certain signals. -Reversible non-covalent modulation - allosteric modulators -small metabolites or cofactors -Reversible covalent modification -inhibition with separate regulatory protein -proteolytic cleavage
Allosteric enzymes -Homotropic -Heterotropic
Heterotropic Allosteric Modulation In multienzyme pathways the regulatory enzyme is inhibited by the end product. Eg., L-Threonine to L-Isoleucine in bacteria.
Kinetics of Allosteric enzymes -sigmoid curve is observed -substrate concentration is represented by [S] 0.5 or K 0.5 homotropic allosteric enzymes
-reflects cooperative interaction between protein molecules -small changes in conc. of modulator can be associated with large changes in activity
Heterotropic Allosteric enzymes -Activation may cause increased velocity for fixed K 0.5 -Negative modulator cause decreased velocity for fixed K 0.5
-increased V max with little change in substrate
Learning check! Match the following Competitive Inhibiton decreases Km and Vmax Uncompetitive inhibition decreases [E]T Mixed inhibition increases Km Inactivation decreases Vmax, while Km may increase or decrease Mixed inhibition is characterized by two dissociation constants for the inhibition. True False
Reversible covalent modification
How do the modulators act? -oxygen atom of phosphoryl group undergo H-bond formation -repulsion of neighboring residues with negative charges phosphatase kinase
Glucose-6-phosphate ATP synthesis in muscles Free glucose in liver
-phosphorylation occurs in structural motif called consensus sequence -AA sequence is not the only factor which determines phosphorylation
Regulation by proteolytic cleavage -zymogen an inactive precursor is cleaved to form the active enzyme
-precursors are also called proprotein eg., collagen, fibrin, and thrombin
Enzyme inhibitors Interfere with catalysis, slowing or halting enzymatic reactions. -Reversible Inhibition -competitive Since it is reversible so increasing the concentration of substrate remove inhibition.
Uncompetitive inhibition Lower the measured V max Apparent K m also decreases
Irreversible Inhibition DIFP Diisopropylfluorophosphate Suicide inactivators such compounds are inactive untill they bind active site of a specific enzyme.