Unit 7: Enzymes
- Enzymes do not change equilibrium - they change kinetics.
- Binding forces holding an enzyme and substrate together can be ionic, electrostatic,
hydrophobic, or some combination. Covalent bonds are less common because they require
more energy and release is more difficult.
- Potential energy is stored in bonds, enzymes work to reconfigure bonds to get them to the most
stable conformation and you get the potential energy out.
- Transition state activation energy between relatively unstable molecule and the more stable.
- G Free energy.
- Stability difference is G. Size of the mountain that must be overcome is difference between G
and transition state (‡). Backwards hill is starting from the product going to reactant, but
typically the hill will be bigger.
- Transition state is distorted and doesn’t look like either of the
reactant or products.
- Catalyst will make the reaction go faster by PROMOTING the
transition state, or LOWERING the activation energy.
Q: how is this catalyst lowering the energy?
o Provide a surface for binding.
o Catalyses hydrogen by added onto double bounded
carbon by sticking to the surface. (versus floating around
o Entropy is reduced, they are not in three dimensions they
are in two. Much more likely to find each other.
o Promoting bond formation by making things less spread
out and by promoting the transition state.
- Enzymes are protein catalysts (2/3) (no metal at all, just collections of amino acids). Amino acid
residues are brought together and interact with the substrate to directly lower transition state.
Have sites that bind and react and provide some kind of surface that catalyzes the reaction.
- Sometimes an active site can bind more than one substrate. Because enzymes work by contact,
you can narrow down where the active site must be.
- EX: Enzyme ribonuclease binds the nucleotide uracil using hydrogen bonds. Specificity: how
does it bind? Forms 3 H bonds to uracil. The enzymes ability to bind to a substrate and only that
substrate is specificity.
- Enzymes can also use large metal atoms (1/3 of enzymes). EX. Cu for cytochrome oxidase.
- Metal ions can be held by organic/metallorganics.
- Coenzymes: unique structure that helps the enzyme work by making the “active site” more
chemically reactive. Most are vitamins; whenever the cofactor is not a metal (NAD)
- Cosubstrate: a noncovalently bound coenzyme
- Prosthetic group: if the cofactor is covalently bond (literally stuck on)
- Holoenzyme: an enzyme with its cofactors bound (whole)
- Apoenzyme: an enzyme without its cofactors (empty)
1. Oxidoreductases: move electrons around (hydride ions or H atoms) (oxidation reduction)
2. Transferases: Group transfer reactions
3. Hydrolases: hydrolysis reactions (transfer functional groups to water)
1 4. Lyases: create double bonds by removing groups. Elimination of functional groups (usually H+
and O- to form a C=C double bond
5. Isomerases: Add isomerase. Transfer groups within molecules to yield isomeric forms.
6. Ligases: Forming bonds by condensation. Requires ATP hydrolysis to provide the energy
- EC 1 number: the kind of reaction catalyzed by the enzyme (above)
- EC 2 rdmber: (1 subcndss) the atoms or bonds acted on by the enzyme
- EC 3 number: (2 subclass) the enzyme type which catalyzes the specific reaction on the
specific type of atom or bond
- EC 4 number: Serial number for each enzyme in subclass 2.
NOTE: type of reaction is what happens, reaction mechanism is how it happens.
1. Acid-base catalysis
2. Covalent catalysis
3. Metal ion catalysis
4. Proximity and orientation effects
5. Preferential binding of transition state complex
General Acid-Base Catalysis / Specific Acid-Base Catalysis:
- If an enzyme moves protons around, it takes place in acid base catalysis. Proton transfer lowers
the free energy of a reaction’s transition state.
- Acid form: has a proton on it (donor). Base form: accepts a proton
- Side chains of amino acids Asp, Glu, His, Cys, Tyr and Lys have pK’s near physiological pH that
allows them to acts as acid/base catalysts.
- The catalytic activity is sensitive to pH which influences the protonation of side chains.
Ex. RNase A acts as a proton donor and proton acceptor in cleaving RNA for hydrolysis.
- Accelerates reaction rates by forming catalyst-substrate covalent bond.
- Enzyme has a nucleophile that attacks the substrate, forming a covalent bond with electrophile.
- Always two steps. Enzyme must be unchanged, therefore you must break the covalent bond and
release the other half of the molecule.
- General hydrolysis reaction: Enzyme attacks one of the two sides of the bond.
Ex. Acetoacetate to acetone via Schiff base intermediate
Metal Ion Catalysis:
- Transition metal ions within the active sites of enzymes are strongly bound to the enzyme. Alkali
and alkaline earth metal ions are more loosely bound
- Metal ions orient a substrate within the active size or neutralize a negative charge on the
- Mg and P are the perfect match because their charges fit together. Mg fits between minuses on
- Metals that can undergo redox changes (Mn and Fe ) act as redox catalysts.
EX. Carbonic anhydrase uses zinc to hold a water molecule in attack position.
o Zinc binds N (three arranged in just a way to bind the Zn enzyme). CO can reac2 with H O 2
Proximity and Substrate Orientation Catalysis:
2 - Important for biomolecular reactions.
- 3D nature of active site allows two substrates to be oriented properly and brought into closer
proximity than possible in bulk solution allows it to occur more efficiently than when contact
- When a charged substrate is guided to the active site by the charge distribution of the enzymes
surface amino acids
Ex. Anion superoxide interacts with positively charged amino acids on the surface of the enzyme
- An electrostatic interaction occurs between substrate and amino acids within the active site.
- Chemical reactivity of the substrate is enhanced.
Transition State Binding:
- The charge distribution on the substrate complements the charge distribution on the enzyme’s
- The substrate is distorted until it can fit in the active site. This distorted substrate is the
Ex. Lysozyme cleaving polysaccharides.
- Change G value from G‡(uncat) to G‡(cat)
- Conversion step conversion is turned into product. Catalyze implies that there are three steps
- Enzyme binds to substrate, substrate goes to product (transformation or actual catalyses),
enzyme is released from product.
- Activation energy for G‡(catalyzed) must be LESS THAN < G‡(uncatalyzed)
- Three steps: E + S ES EP E + P
- Enzyme does not affect the overall equilibrium. (S P) Kinetics are what is affected by the
enzyme. Does not change HOW the bonds rearrange, rather how quickly they rearrange.
- Catalysis only works in the middle, it does not work in the ends. The ground states of reactants
and products do not change, only the transition states
- Affect RATES not EQUILIBRIUM
- V: velocity of an equation
- k: rate constant. tells you the rate, but is NOT the rate itself
- First order kinetics: For S P: V = k[S]
o By varying concentrations you determine if it is first order or not
- Second order kinetics: For S +1S 2 P: V = k[S1][S2]
- G‡ is in k per molar units? you can directly convert the two. Another way of saying the rate
constant. (logarithmic relationship) k will change a lot more than G
- Rate enhancement: how much the k changes.
Q: how to enzymes change the transition state?
- They bind to the substrate, product and transition state. Will have a certain affinity for both, but
also has a certain high affinity for the transition state
3 - Enzymes bind the transition state of the reaction to make it lower in energy stabilize it and
make it more likely to happen (thus reaction more likely to happen).
- An enzyme that binds it substrate too well never changes the substrate, it ends up being a
- Something that is too complementary to its substrate will make it harder for the substrate to
continue on to the product.
- Myoglobin sticks to oxygen and sits there, doesn’t do anything (will never change the
- Enzyme must be complementary to transition state. Able to bind weakly at substrate, that pulls
the substrate to the transition state (and beyond).
- EX: DNA binding proteins. A protein that binds DNA but does not cut it binds as a straight
helix in its most stable form.
- DNA endonucleases cut DNA after bending it. Cuts the DNA right near the middle of the bend.
- Binding the transition state stabilizes it and lowers the G‡
- If you take the differences in k you get numbers around 60-100 kJ/mol needed for normally
observed enzymatic rate enhancements.
- Enzymes can also work just by bringing things together.
- Binding two things in the right orientation.
- If you connect the two ends, you get a significant increase in rate. The two molecules can rotate
but they are stuck together. Reducing the entropy/chaos.
- You are not activating the groups
- Reduce entropy/rotation further and the rate increases even more. Make the other ends more
“solid” by decreasing potential rotations
- Some enzymes move when the substrate binds. The substrate bends the enzyme around itself.
- Some enzymes are not flexible and have a structure binding site to match the substrate lock
- stripping water away to bind something
- Water is around the substrate, and when it binds the surface of the enzyme and substrate must
be desolvated before they can bind.
- Allows the hydrophobic effect to work.
- Curve will never follow a straight line, the reaction will eventually even and flatten out. Slope
starts off large positive, and then changes to zero. CONSTANTLY CHANGING.
- When you mix 1uM substrate with enzyme and time how fast product is produced you get an
initial line that starts to curve.
- The lower the substrate, the slower the rate of reaction, and the less product you will have at
equilibrium. (Equiilibrium changes depend on [S])
4 Steady State:
- In normal enzyme kinetics, the pre-steady state happens too fast to measure