Biochem Exam 3 Stuff

Enzymes

The workers of the cell

Enzymes are proteins that carry out a chemical reaction (Note that RNAs can also do that!)

Enzymes act as catalysts of chemical reactions so the reactions occur at a much faster rate than uncatalyzed reactions

As we will see, they do so through two mechanisms:

• By providing a protected environment

• By providing all of the reactive partners [acids (Asp, Glu, C-term), bases (Lys, Arg, His, N-term), and hydrogen-bonding] in the correct configuration

General Properties of Enzymes:

• Higher reaction rates

10⁶ - 10¹² times greater than those of uncatalyzed reactions and at least ~10⁷ times greater than chemically catalyzed reactions

General Properties of Enzymes:

• Milder reaction conditions

• Near neutral pH, < 100°C, atmospheric pressure

• Chemical catalysis often requires high T, pressure, and pH

General Properties of Enzymes:

• Greater reaction specificity

Enzymatic reactions are generally highly specific for substrates and therefore rarely produce any side products

General Properties of Enzymes:

• Capacity for regulation

Enzymatic catalysis can be regulated

• Allosteric control, covalent modification, and variations in synthesis of the protein (i.e. protein expression)

Geometric and electrostatic complementarity (h=hydrophobic) between enzyme and substrate is…

The basis of a “lock and key” model of enzyme function

Many substrate binding pockets are pre-formed,…

Some adjust to accommodate substrate – this is called “induced fit”

Enzymes are stereospecific

Aconitase converts the pro-chiral citrate to the chiral isocitrate in the citric acid cycle

Aconitase can distinguish between the two acetate groups and thus produces only one enantiomer of isocitrate

This is because ligand binding sites are chiral and highly stereospecific

Preferentially or exclusively bind one enantiomer and…

Carries out stereospecific reactions

Recall that enzymes are constructed of chiral amino acids

These chiral amino acids create a chiral substrate binding pocket

Even when the substrate of a reaction is not chiral, catalysis proceeds stereospecifically and…

Often leads to a chiral product

L-proteins act on specific chirality, therefore…

D-proteins will act on opposite chirality

Cofactors

Molecular modules that enable enzymes to expand their repertoire of chemistry!

Metal ions

Essential non-protein elements of some enzymes

• Cu²⁺, Fe³⁺, Zn²⁺, etc.

Coenzymes

Organic/biological cofactors

• Cosubstrates & Prosthetic groups

Cosubstrates

Transiently enzyme-associated coenzymes

• NAD⁺, NADP⁺, etc.

Prosthetic groups

Permanently enzyme-associated coenzymes

• Heme, pyridoxal phosphate, etc

Coenzymes are generally reduced/oxidized during a reaction and must be returned to their initial state

This can be done by the same enzyme or a separate one

Holoenzyme

Catalytically active enzyme-cofactor complex

Apoenzyme

Inactive protein resulting from removal of cofactor

Apoenzyme (inactive) + cofactor ↔ …

Holoenzyme (active)

Activation Energy and Reaction Coordinate

A + B ⇌ X^‡ → P + Q

Reaction coordinate

Reactants must approach one another along the path of minimum free energy

The reaction coordinate will have one or more transition states which represent free energy

The reaction coordinate starts with the reactants, goes through one or more transition states, leading to the product

Transition State Diagram; Reaction:

• A&B: reactants

• X^‡: the transition state

• P&Q: the products

ΔG^‡ is the activation free energy

ΔG_reaction is the overall free energy of the reaction

The greater ΔG^‡…

The slower the reaction rate

Rate-limiting or rate-determining step

When one reaction step is much slower than all the rest this step acts as a “bottleneck”

I = intermediate

Highest activation energy is rate-limiting

Catalysts (and enzymes) reduce the activation free energy of a reaction, both in the forward AND reverse directions

Enzymes cannot change the ΔG_reaction!!!!

Catalytic Mechanisms; Enzymes use multiple different mechanisms to catalyze biochemical reactions:

1. Acid-base catalysis

2. Covalent catalysis

3. Metal ion catalysis

4. Electrostatic catalysis

5. Proximity and orientation effects

6. Preferential binding of the transition state complex

Acid-Base Catalysis

Mechanism of keto-enol tautomerism

• a) Uncatalyzed

• b) General acid

• c) General base

AA can act as general base/acid

His12 abstracts 2’-proton, 2’-O^- nucleophilically attacks phosphate causing other oxygen to grab a proton from His119

His12 acts as a general base and His119 as a general acid (step 1)

His12/His119 then switch roles (step 2)

His12 and His119 are restored (end)

Covalent Catalysis

Covalent catalysis involves the formation of a transient covalent bond between the catalyst and the substrate

Decarboxylation of acetoacetate:

• Uncatalyzed at top

Catalyzed reaction at bottom

Nucleophilic catalysis:

• Substrate forms a bond with enzyme/coenzyme

RNH₂ is from the enzyme or cofactor

Covalent bond is transiently formed, …

Accelerating the reaction

Carbonic Anhydrase Reaction

It converts between CO₂ and HCO₃^-

HO^- is stabilized by Zn²⁺ where it nucleophilically attacks CO₂

A water molecule completes the process, regenerating the cofactor and liberating the product

Electrostatic Catalysis

• Bulk water is often excluded from enzymatic active sites

This leads to an organic-like environment in the active site

In this environment, electrostatic interactions are more important

i.e. since they are less shielded – lower dielectric constant

The pKa’s of ionizable amino acids can be substantially shifted from their normal values in the protein environment

For example, the HIV protease has two Asp residues near one another in the active site. They share a single proton – the net charge on this pair of Asp residues is –1 (R-COO^-…H⁺…-OOC-R). In a typical protein, both Asp residues would have a full –1 charge.

In addition, the charge distribution in active sites is often preferentially arranged so as to stabilize the transition state, potentially guiding the polar substrates towards…

The binding site (electrostatic complementarity) and leading to a lowering of the activation barrier

Uncatalyzed (red) and enzymatically catalyzed (blue) reaction

• Note the minimum for the enzyme-substrate (ES) complex

• It’s called the Michaelis complex

Note that the substrates and product have the same energy in the catalyzed versus uncatalyzed

However, the ES^‡ is preferentially bound and stabilized

This is what is responsible for the enhancement in catalytic rate

Enzymes may bind the transition state of the reaction it catalyzes with greater affinity than its substrates or products!

Enzymes typically stabilize the transition state of a reaction more than that of the reactants or the products

This leads to the idea of small TS-like molecules that might act as “inhibitors” of the enzymatic process by more strongly binding to the active site than the substrate