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what are enzymes?

Catalysts that carry out biochemical reactions


what is a catalyst?

a substance that changes the rate of a chemical reaction without affecting the position of the equilibrium or being changed during the course of the reaction.


*how do enzymes work as biological catalysts?

keep in mind that most biochemical reactions will not proceed at any rate in the absence of an enzyme; they can increase the rates of biochemical reactions by 10^4 to 10^10; generally no side reactions; chemical catalysts catalyze a number of unwanted side reactions.


are enzyme catalyzed reactions fully reversible?

yes, most of them


do enzymes have an active site?



where do substrates bind in relation to the enzyme?

the binding site, a cleft of the protein, also known as the catalytic or active site of the enzyme


does catalysis involve amino acids? if so which are the common amino acids used?

yes; catalysis involves the actions of one or more amino acid residues with reactive side chains like ser, cys, his, glu, asp specifically located near the substrate binding site


enzymes are not highly specific, T/F?

F, they are highly specific; they often react with only a single molecule and even its stereoisomers are ignored to give stereo specific products, a chemical catalyst usually causes a loss of stereochemistry


what is catalytic activity dependent on?

the tertiary structure of the enzyme, one enzyme one function


*what are the factors that affect enzyme activity?

substrate concentration, temperature and pH


*how does substrate concentration affect enzyme activity?

the rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity is achieved (Vmax); leveling off indicates saturation of all binding sites


*how does temperature affect enzyme activity?

reaction velocity will increase with temperature until a peak velocity is reached (more molecules have inc. energy to pass over barrier); further elevation of temperature causes a decrease in reaction velocity due to denaturation of enzyme; so in short physiological temperature does not vary significantly so this is not a viable means to control enzyme activity


*how does pH affect enzyme activity?

it effects ionization state of the active site, extreme pH leads to denaturation of the enzyme though pH at which maximal enzyme activity is achieved is different for each enzyme and reflects the (H^+) at which the enzyme functions; pH can affect protein folding and 3D structure but also specific amino acid side chains located inside the active site


*what are the different types of enzyme classes?

oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, isozymes


*what is an oxidoreductase?

add or remove one or more electrons from or to substrates and are sometimes called dehydrogenases because their oxidation reactions often remove two electrons and a proton from the oxidized substrate


*what is a transferase?

transfer a group from one substrate to another


*name an example of a transferase?

kinase, transfer phosphate groups from ATP to substrate


*what are hydrolases?

carry out the hydrolysis of substrates


*name an example of hydrolase?

phosphatase which remove phosphate groups from substrates


*what is a lyase?

carry out the cleavage of a molecule into parts


*what is an isomerase?

rearrange a substrate into an isomeric form (i.e., D to L form)


*what is a ligase?

join molecules together by condensation, usu. eliminating water.


what is an isozyme?

different enzyme that carry out the same reaction but with different regulatory properties or in different locations within the organism (liver vs. heart, etc)


what is the free energy change for a chemical reaction?

it determines the spontaneity of a reaction like which direction a reaction is likely to proceed


*what is the change in Gibbs free energy (ΔGo’) for a reaction?

it is the difference in energy between the products and the substrate or the free energy change under biological conditions


*what happens if the ΔG is negative?

the reaction will proceed spontaneously with the release of energy


*what happens if the ΔG is positive?

the reaction will not proceed spontaneously


*what happens if the ΔG = 0?

the reaction is at equilibrium and although the substrates react to form products and products react to form substrates there is not net change in concentrations


*enzyme catalysis can change both the rate at which the equilibrium in the reaction is achieved and position of the equilibrium (thermodynamics), T/F?

false, the position or thermodynamics of the reaction cannot be changed


*explain what happens in an energy coordinate diagram for an enzyme catalyzed reaction, because you will have to know how to interpret it?

in an energy coordinate diagram, in order to proceed with a chemical reaction you have to change the electronic structure of the bond in the substrate (enzyme binding with substrate to illicit a change in structure) to raise it to a transition state. Once there, a decision has to be made if the molecule wants to go back to its initial state or move forward breaking bonds producing a carboxylic acid and a free amino group. The forward reaction is favored because the final energy is much lowered and also energy is given away, producing a negative delta G or being spontaneous


enzymes act on substrates changing it from the initial form to the final form, T/F?

false, enzymes increase the rate of a reaction by decreasing the reaction activation energy


what is the rate of the reaction dependent on?

the magnitude of the activation energy, E sub A.


what does K sub eq (equilibrium constant) tell you?

how much product and reactant there is and is written product/reactant and so if we relate this back to ΔGo’ it tells us something about the equilibrium and vice versa


the binding of the enzyme to substrate forms weak interactions and energy is liberated used to decrease the activation energy to achieve lower state, T/F?



what are the four ways enzymes increase the rate of a reaction?

binding energy, proximity, induced fit, orientation


what is binding energy?

interaction of S with enzyme active site providing binding energy, bringing S closer in structure to the transition state. This lowers the activation energy and increases the reaction rate


what is proximity?

Binding substrates increases their effective concentration within the active site, thereby increasing the reaction rate.


what is induced fit?

Binding of S induces changes in the tertiary structure of E (conformational changes) that in turn distort S toward the transition state.


what is orientation?

geometry of S binding the active site will allow the reacting molecules to be in the perfect orientation for reaction, thus increasing the reaction rate.


Recall the terms “binding energy,” “proximity effect” and “induced fit” and relate these terms to an enzyme’s ability to lower the activation energy of a reaction.

so enzyme recognizes substrate and they are brought together in a proximity effect so they are one bond distance apart and can react. Keep in mind too about orientation so that the atoms of each substrate and enzyme are right by each other and of course you also have induced fit once you have substrate enzyme binding and conformation change occurs


what is also known as the catalytic triad?

histdine, serine and aspartate


what does serine protease do?

a family of enzymes that hydrolyze specific peptide bonds in proteins (i.e., splits the protein into smaller polypeptides) and these enzymes hydrolyze amide bonds adjacent to specific amino acid residues. The main area is digestion and these are synthesized in the pancreas as pro-enzymes or zymogens that are initially inactive and become active after they are released from the pancreas


how do these proteases work?

so these proteases or enzymes recognize a specific amino acid and so if you take chymotrypsin for example, it will break a peptide bond beside an aromatic amino acid and so you see a hydrophobic binding pocket favoring the aromatic residues and large nonpolar residues and so in short the process is used for identification purposes other examples include trypsin and elastase

The purpose of the pocket is to bind the substrate protein side chain and hold it in place, so that the active site of the protease can cut the peptide bond on the substrate protein


what type of reaction mechanism does serine protease perform?

This is a great example of the proximity effect and also orientation: the individual amino acids that make up the catalytic triad are far apart in the primary sequence, but are right next to each other in the tertiary fold. Thus they can interact to provide an activated oxygen atom on the serine. Plus, the specificity pocket that binds the Tyr residue positions the substrate peptide perfectly in the active site so that the target peptide bond is right beside the activated serine oxygen. Together, this facilitates a highly specific and rapid reaction.


how does the catalytic triad tie into the serine protease reaction mechanism?

The “catalytic triad” of residues cooperate to make the Ser-OH a strong nucleophile, which attacks the peptide carbonyl bond allowing for proper orientation and proximity effect (for both enzyme and substrate and amino acids with it) for reaction to take place and transition state to provide for stabilization, though it can go forward weak interactions are holding the substrate in such a way compelling the reaction to move forward and so once formed, the transition state breaks down to more stable products and the target peptide bond is cleaved. The first product (a peptide piece) is released, whereas a fragment remains bound to the enzyme. The enzyme-substrate bond is broken by hydrolysis. The enzyme interacts with water, making it a stronger nucleophile, which attacks the carbonyl carbon.
Think proximity and orientation


why don't proteins break down spontaneously in the cell?

the serine is in position to bind with carbon that allows that decision to be made to move forward and isn't bound to a carboxyl group


how does aspartyl protease work?

activation of water for nucleophilic attack, no enzyme-bound intermediates, rearrangement of tetrahedral carbon intermediate leads to cleavage of peptide bond. This is what we would see with viral proteases and they use this enzyme to carry out their function, HIV for example


what is the binding pocket like for chymotrypsin?

a hydrophobic binding pocket that favors aromatic residues and large non-polar residues


what is the binding pocket like for trypsin?

a deep binding pocket with a negatively charged residue (glu) at the bottom that favors the binding of lys and arg


what is the binding pocket like elastase?

a very small binding pocket that favors binding of ala or gly residues.


what is the purpose of the binding pocket?

The purpose of the pocket is to bind the substrate protein side chain and hold it in place, so that the active site of the protease can cut the peptide bond on the substrate protein


so we know that enzymes decrease the activation energy of a reaction and that weak interactions are formed with substrate, liberating energy used to lower the activation energy, how?

Any time a weak interaction is formed (H-bond, ionic interaction, hydrophobic interaction, van der Waals), a tiny bit of energy is released. The enzyme will capture the sum of all these small energy changes and use this energy to lower the energy required to reach the transition state.


how is the equilibrium position determined?

the free energies of substrate and product


explain the principles of catalysis?

1) substrate binding

2) histidine activates serine for nucleophilic attack

3) the oxyanion tetrahedral intermediate is stabilized by hydrogen bonds

4) cleavage of the peptide bond

5) the covalent acyl-enzyme intermediate

6) water attacks the carbonyl carbon

7) second oxyanion tetrahedral intermediate

8) acid catalysis breaks the acyl enzyme covalent bond

9) product is free to dissociate