Lecture 9 Flashcards
Enzyme Mechanisms
Big Picture Items
- Enzymes catalyze reactions in many small steps
- Enzymes can use a wide variety of methods for catalysis
- An enzyme can use a mix of methods
- Enzymes lower the free energy of the transition state (TS)
- Enzymes can be highly specific regarding the characteristics of a substrate or show much less specificity
- Serine proteases contain a characteristic “catalytic triad”
- The same catalytic triad is found in enzymes with entirely different folds: “convergent evolution”
Catalysis by Enzymes
Enzyme catalysis can :
• be extremely substrate specific, or quite substrate-unspecific
• be very fast, or quite slow
• occur with almost no, or with large conformational changes
• act on one substrate, or multiple substrates
• be quite pH-dependent
• involve only protein side chains, or involve also very complex “co-factors”
Enzyme Names
Enzymes are usually named by appending “ase” to the name of the substrate, or one of the substrates.
Reaction Coordinate
A + B –> P + Q
The “reaction coordinate” is the path of minimum free energy G during the reaction.
The highest point along this path is the “transition state” of the reaction.
The configuration of substrate(s) at this point is the “transition state” X‡.
The G of X‡ is the free energy of the “transition state” of the reaction.
This ΔG‡ is called the free energy of activation of the reaction.
Reaction Rate Acceleration
By “Transition State Stabilization”
G of black X‡ is the free energy of the ”transition state” of the uncatalyzed reaction
Enzymes speed up reactions by providing a reaction pathway whose free energy of activation is lower, by ΔΔG‡cat , than that of the uncatalyzed reaction.
The larger the absolute value of ΔΔG‡
cat, the better the enzyme.
Catalytic Mechanisms in Biology
Enzymes use the following mechanisms,
and often a combination of these:
A. Acid-Base Catalysis
B. Metal Ion Catalysis
C. Catalysis via Proximity & Orientation
D. Covalent Catalysis – nucleophiles & electrophiles
E. Catalysis by Preferential Transition State Binding
Biological catalysts (enzymes and ribozymes),
and chemical catalysts, follow the same principle:
they lower the activation energy barrier to speed up the reaction.
CATALYSTS, HENCE ALSO ENZYMES,
DO NOT AFFECT THE EQUILIBRIUM OF A REACTION
Acid-Base Catalysis
REACTIONS INVOLVING PROTON TRANSFER
• General Acid Catalysis: general acid donates a proton to the substrate
Enzyme Active site functional group (e.g., amino acid) must be protonated
• General Base Catalysis: general base accepts a proton from the substrate
Enzyme Active site functional group (e.g., amino acid) must be deprotonated
• Concerted Acid-Base Catalysis:
a general acid and a general base both participate in the reaction
A. RNases
A very important class of enzymes (using acid-base catalysis)
Rnase Nomenclature:
Endonucleases: cleave in the middle
Exonucleases: cleave at either end.
RNase A:
An endonuclease which cleaves the P-O bond shown in blue and indicated with the blue arrow.
Sequence specificity: RNase A does not have much nucleotide sequence preference. This makes sense since the enzyme has to cleave RNA in the digestive tract into smaller pieces.
Ribonuclease A
Ribonuclease A (RNase A) is an endonuclease that cleaves single-stranded RNA
(Actually, shown is Ribonuclease S, consisting of S-protein and S-peptide. But this is irrelevant for catalysis)
An all-β protein with four disulfide bridges.
A non-hydrolysable dinucleotide substrate analog (red) is bound in the active site.
Two histidine imidazoles (green) are ready to catalyze the hydrolysis if this were RNA.
Ribonuclease A Catalysis
- His12 acts as a general base abstracting a proton from the 2’-OH of the ribose.
- The 2’-OH carries out a nucleophilic attack on the adjacent phosphorus atom of the RNA.
- His119 acts as a general acid donating a proton to the 5’-OH of the leaving ribose.
- A 2’,3’-cyclic intermediate is formed.
- The first product leaves.
- Water comes in near His119.
Ribonuclease A Catalysis
- His119 acts as a general base abstracting a proton from the water.
- The water OH carries out a nucleophilic attack on the phosphorus atom of the intermediate.
- His12 acts as a general acid donating a proton to the 2’O of the leaving ribose.
- The second product is formed.
B. Metal Ion Catalysis
The enzyme “Carbonic Anhydrase” uses Zn2+ to catalyze the reaction:
- The Zn2+ polarizes a water molecule which ionizes due to the action of a 4th Histidine
(not shown) and becomes OH-. - The OH- performs a nucleophilic attack on the C atom of the CO2 substrate.
- The HCO3- product forms and leaves.
- The next water comes in, and back to step 1
The active site of human carbonic anhydrase
Three imidazole side chains of histidines are exquisitely positioned to bind the zinc-ion such that the ion can still bind a water molecule.
C. Proximity and Orientation Effects
Bringing reactive groups in close proximity and in the proper orientation can accelerate reactions more than a million fold!!
As seen in this reaction forming an anhydride.
The catalysis of peptide formation by the ribosome is an example of catalysis by proximity and orientation.
D. Covalent Catalysis
• In covalent catalysis, a covalent bond is transiently
formed between the substrate and the enzyme (or coenzyme)
• This reaction usually involves a nucleophilic group on the enzyme and an electrophilic group on the substrate
Nucleophiles and electrophiles
- Biological nucleophiles (shown on blue shadow on the left) are negatively charged or contain unshared electrons. (Note: the oxygen of water can also act as a nucleophile).
- Biological electrophiles (shown in red on the right) are either positively charged, contain unfilled valence shells, or contain an electronegative atom.
