A protein with catalytic properties due to its power of specific activation How do enzymes catalyze chemical reactions? Theoretical concept: transition state theory Catalytic mechanisms: • Acid-base catalysis • Covalent catalysis • Metal ion catalysis • Electrostatic catalysis • Catalysis through proximity and orientation effects • Catalysis by preferential transition state binding Example: Serine proteases Transition state Making reactions go faster
Increasing the temperature makes molecules move faster Biological systems are very sensitive to temperature changes. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy. They create a new reaction pathway “a short cut”
An enzyme controlled pathway Transition state theory Transition state diagrams illustrate the path of a reaction The transition state - the point of highest energy, i.e. the most unstable situation ΔG‡ is the difference of energy of the reactants and the transition state (free energy of activation) ΔG‡ determines the rate of the reaction
The reaction rate is proportional to e- ΔG‡/RT R is the gas constant and T the absolute temperature (Eyring equation) greater the value of ΔG‡, the slower the reaction rate Acceleration of a reaction rate can therefore be achieved by lowering ΔG‡ Enzymes reduce ΔG‡ Catalysts lower the energy of activation of a chemical reaction and hence accelerate the rate of reaction The rate of a chemical reaction can be accelerated by up to 1017-fold The rate acceleration is enzyme specific and depends on the nature of the chemical reaction. a catalyst does not change the equilibrium constant between reactants and products Acid-base catalysis A reaction is acid-catalyzed if proton donation (a Brønsted acid) lowers the free energy of activation and leads to an acceleration of the reaction rate.
A reaction is base-catalyzed if proton abstraction (a Brønsted base) lowers the free energy of activation and leads to an acceleration of the reaction rate.
Reactions catalyzed by the concerted action of a proton donation and abstraction are called acid-base catalyzed. Acid or base catalysis of keto-enol tautomerism RNAase A - example for an enzyme utilizing acid- base catalysis Function: Hydrolysis of RNA to component nucleotides
His12 abstracts a proton from the 2’-OH group. This promotes the nucleophilic attack on the phosphorus resulting in P-O bond cleavage. His119 acts as a general acid by protonation of the oxyanion leaving group. This results in the formation of a 2’,3’-cyclic intermediate which can be isolated under certain conditions. Water is not admitted to the active site and drives essentially the reversal of the initial process leading to complete hydrolysis. Covalent catalysis Catalyst forms a transient covalent linkage with the substrate leading to rate enhancement. Can be divided into two phases: 1. Nucleophilic reaction between the catalyst and the substrate to form a covalent bond 2. Withdrawal of electrons from the reaction center by the electrophilic catalyst (e.g. hydrolysis of the Schiff base).
Note that in cases where the nucleophilicity is the rate-determining step, the reaction rate tends to increase with the basicity (pK) of the catalyst. Covalent catalysis Top: uncatalyzed decarboxylation of acetoacetate
Bottom: primary amines as catalyst for the decarboxylation of acetoacetate
Groups involved in covalent catalysis Among the amino acids, the following side chains can be used for covalent catalysis:
• Serine: hydroxyl group (an example will follow later!) • Cysteine: thiol group • Histidine: imidazol ring • Aspartate & glutamate: carboxyl group • Lysine: amino group (Schiff base formation)
In addition, enzymes can utilize organic compounds, so called coenzymes, such as pyridoxal phosphate and thiamine pyrophosphate (vitamins!) for covalent catalysis. Metal ion catalysis
One third of all enzymes require the presence of metal ions for activity!
1. Metalloenzymes - contain tightly bound metal ions, most commonly transition metals such as iron, copper, manganese and cobalt.
2. Metal-activated enzyme - bind metal ions loosely from solution, most commonly alkali and alkaline earth metals such as sodium, potassium, magnesium and calcium Roles of metal ions in catalysis
• Bind to substrate in order to properly align it for catalysis
• Mediate oxidation-reduction processes (redox biochemistry) through reversible interchange of the metal ion’s oxidation state.
• Stabilization/shielding of negative charges during the catalytic process. Metal ions promote nucleophilic catalysis
Metal-bound water exhibits a much lower pKa value The resulting metalbound hydroxyl group can act as a potent nucleophile
The essential zinc in the active site is bound by three histidine side chains. Water occupies the fourth binding site and is polarized by the zinc atom. The bound water deprotonates and the resulting hydroxyl attacks carbon dioxide converting it to carbonate. Proximity and orientation effects Binding of the substrate facilitates catalysis in three ways:
Proximity - effect is contributing a rate acceleration of ca. 5-fold Orientation - Reaction rates are accelerated approx. 100- fold by this effect Freezing out motion - the enzyme restricts the translational and vibrational freedom of the substrate an „prepares“ it for a transition state like structure. The rate enhancement achieved in the order of 107 The active site For catalysis
The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily
Cofactors
An additional non-protein molecule that is needed by some enzymes to help the reaction Tightly bound cofactors are called prosthetic groups Cofactors that are bound and released easily are called coenzymes Many vitamins are coenzymes
Types of Cofactors
Coenzyme: The non-protein component, loosely bound to apoenzyme by non-covalent bond. Examples : vitamins or compounds derived from vitamins. Prosthetic group - The non-protein component, tightly bound to the apoenzyme by covalent bonds.
Some enzymes require cofactors To take over chemical reactions that cannot be performed by amino acid side chains Required in diet of organisms Organic molecules can associate with enzyme as cosubstrate (NAD+) The structure and reaction of NAD+ NAD+ obligatory cofactor In alcohol dehydrogenase reaction
NADH dissociates from the enzyme to be re-oxidized in an independent reaction Prosthetic groups Permanently associated with enzymes By covalent bonds Example: heme is bound to proteins called cytochromes Summary Enzymes Transition states Activation energy Acid-base catalysis Covalent catalysis Metal ion catalysis Cofactors/coenzymes/cosubstrates/prosthetic groups
The substrate
The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site
What happens at the active site? Lock and key hypothesis
In the lock-and-key model of enzyme action: -the active site has a rigid shape -only substrates with the matching shape can fit -the substrate is a key that fits the lock of the active site This explains enzyme specificity. This explains the loss of activity when enzymes denature This is an older model, however, and does not work for all enzymes.
Enzymes-how do they work? Induced-fit hypothesis: o When a substrate begins to bind to an enzyme, interactions induce a conformational change in the enzyme o Results in a change of the enzyme from a low catalytic form to a high catalytic form o Induced-fit hypothesis requires a flexible active site
(b)
Catalysis in the Enzyme’s Active Site
In an enzymatic reaction The substrate binds to the active site Held by weak interactions (hydrogen bonds) Side chains (R) from amino acids catalyze the conversion of substrate to product Product leaves the active site Enzyme is free to take another substrate molecule into its active site Cycle happens very fast Metabolic reactions are reversible Enzyme can catalyze both forward and reverse reactions Enzyme catalyzes the reaction in the direction of equilibrium Use variety of mechanisms to lower the activation energy and speed up a reaction The catalytic cycle of an enzyme
1 Substrates enter active site; enzyme changes shape so its active site 2 Substrates held in embraces the substrates (induced fit). active site by weak interactions, such as hydrogen bonds and ionic bonds.
3 Active site (and R groups of Substrates Enzyme-substrate its amino acids) can lower EA complex and speed up a reaction by • acting as a template for substrate orientation, 6 Active site • stressing the substrates Is available for and stabilizing the two new substrate transition state, molecule • providing a favorable microenvironment, Enzyme • participating directly in the catalytic reaction.
5 Products are Released. 4 Substrates are Converted into Products Products. Enzyme Inhibitors
Certain chemicals can inhibit the action of enzymes Inhibitors Attach to enzyme by covalent bonds Usually irreversible
Enzyme Inhibitors Many enzyme inhibitors bind by weak bonds In that case inhibition is reversible Some reversible inhibitors resemble the normal substrate molecule and compete for admission into the active site Competitive inhibitors Reduce the productivity of enzymes by blocking substrates from entering the active site Competitive inhibitors
A substrate can Substrate bind normally to the active site of an enzyme. Active site
Enzyme
(a) Normal binding
A competitive inhibitor mimics the Competitive substrate, competing inhibitor for the active site.
Figure 8.19 (b) Competitive inhibition Noncompetitive inhibitors Do not directly compete with the substrate They impede enzymatic reactions by binding to another part of the enzyme Causing enzyme to change its shape Renders the active site less effective at catalyzing the conversion of substrates to product Noncompetitive inhibitors
A noncompetitive inhibitor binds to the enzyme away from the active site, altering the conformation of the enzyme so that its active site no longer functions.
Noncompetitive inhibitor
Figure 8.19 (c) Noncompetitive inhibition Regulation of enzyme activity helps control metabolism
A cell’s metabolic pathways Must be tightly regulated Controlling where and when various enzymes are active Can be done by switching on and off certain genes that encode specific enzymes Allosteric Activation and Inhibition
Many enzymes are allosterically regulated Have two or more polypeptide chains or subunits Each has its own active site The entire complex oscillates between two conformational states: catalytically active and inactive
Simplest case of allosteric regulation: Activating or inhibiting regulatory molecule binds to a regulatory site (located where subunits join) Binding of activator stabilizes the conformation that has functional active site Binding of inhibitor stabilizes inactive form of the enzyme Subunits fit together so that conformational change in one subunit is transmitted to all others Activator or inhibitor that binds to one site will affect the active sites of all subunits Allosteric activater stabilizes active from Allosteric enyzme Active site with four subunits (one of four)
Regulatory site (one of four) Activator Active form Stabilized active form
Allosteric activater stabilizes active form Oscillation
Non- Inactive form Inhibitor Stabilized inactive functional form active site
(a) Allosteric activators and inhibitors. In the cell, activators and inhibitors Figure 8.20 dissociate when at low concentrations. The enzyme can then oscillate again. Other kind of allosteric activation: If enzyme has multiple subunits, binding (induced fit) of the substrate to one subunit can trigger conformational change in all other subunits Cooperativity Amplifies the response of enzyme to substrates
Cooperativity Binding of one substrate molecule to active site of one subunit locks all subunits in active conformation.
Substrate
Inactive form Stabilized active form
(b) Cooperativity: another type of allosteric activation. Note that the inactive form shown on the left oscillates back and forth with the active Figure 8.20 form when the active form is not stabilized by substrate. Feedback Inhibition
In feedback inhibition The end product of a metabolic pathway shuts down the pathway Some cells use this pathway to sythesize one amino acid from another Prevents the cell from wasting chemical resources
Factors affecting enzymes
All enzymes work best at only one particular temperature and pH: this is called the optimum. Factors that affect the rate of a reaction include: o substrate concentration o pH o enzyme concentration o surface area o pressure o temperature
Different enzymes have different optimum temperatures and pH values.
Factors affecting enzymes
If the temperature and pH changes sufficiently beyond an enzyme’s optimum, the shape of the enzyme irreversibly changes. This affects the shape of the active site and means that the enzyme will no longer work. When this happens the enzyme is denatured. pH and reaction rate
pH also affects the rate of enzyme-substrate complexes Most enzymes have an optimum pH of around 7 (neutral)
Substrate concentraton and reaction rate
The rate of reaction increases as substrate concentration increases (at constant enzyme concentration) Maximum activity occurs when the enzyme is saturated (when all enzymes are binding substrate)
Structure of enzymes
Apoenzyme and Holoenzyme
Apoenzyme is the enzyme without its non-protein moiety and it is inactive. Holoenzyme is an active enzyme with its non-protein component.
Summary Substrate Lock-and-key hypothesis Induced fit Inhibitions Competitive/noncompetitive inhibition Factors affecting enzymes Apoenzymes vs Holoenzyme