Enzymes

Different structural forms of a protein that have the same function (iso = similar or same)

A small Km for an enzyme relfects a high affinity of the enzyme for the substrate

• A Large Km has the opposite effect
• (Km does not vary with the concentration of the enzyme!!)

A high Km means that a lot of substrate is needed to fill half of the enzyme active sites

Directly proportional to the enzyme concentration at any given time

• Reaction rate is independent of substrate concentration: A-B; V= k
• (All of the enzymes are saturated with substrate)

• Reaction rate depends on the concentration of one substrate.
• A - B; V = k x [A]

Reaction rate depends on the concentrations of two substrates.

A+ B - C + D; V = k x [A] x [B]

When there are two substrates but only one is rate limiting.

Example: Hydrolysis reactions in aqueous solution.


A + H2O - C + D; V = k x [A]

• Catalyze oxidation-reduction reactions (NADH)
• an enzyme that catalyzes the transfer of electrons from one molecule to another
• A– + B → A + B–

Catalyze transfer of functional groups from one molecule to another.

Catalyze hydrolytic cleavage

A–B + H2O → A–OH + B–H

Catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement.

Catalyze intramolecular rearrangement

Catalyze reactions in which two molecules are joined (ATP dependent reactions)

• Nonbonding reversible reactions
• E + S ⇌ ES → EP ⇌ E + P

drive endergonic reactions (Delta G > 0) aka. energy is absorbed

the reaction is spontaneous.

the reaction is non-spontanous.

the reaction is at equilibrium

By coupling to a favorable reaction

That means that the reaction is spontaneous

An endergonic and an exergonic that are linked energetically; the endergonic reaction is driven by the exergonic reaction.

Both reactions occur simultaneously and share a common intermediate which cancels out in the final sum.

Biochemical standard (in vitro) free energy change. This is the free energy change when the reactants and products are initially at 1.0M, at 25°C, at 1atm and the H+ concentration is pH 7.0.

Is the actual free energy change in the cell. It depends on the actual concentrations of reactants and products.

Contain only the polypeptide portion

Contain the polypeptide portion & a non-protein portion; need both for activity

The non-protein portion of a conjugated enzyme

The protein portion of a conjugated enzyme

An organic cofactor, or a metol ion (Fe2+ , Mg2+ , Zn2+)

The complete, native, enzyme

An inactive precursor of a native enzyme (not the same as apoenzyme) - part of polypeptide has to be removed to give native enzyme

Co Enzymes

The most common biological handle

Water soluble: located in aqueous environment and/or held in enzyme by polar/ionic forces

Lipid soluble: located in nonaqueous environment and/or held in enzyme by van der Waals forces (hydrophobic force)

Accomplish goals amino acid side chain units can not

acid/base and nucleophilic/electrophilic changes

Reductions and oxidations

Reduced coenzymes (NADH, FADH2)

High-energy phosphate compounds - free energy of hydrolysis more negative (-25 kJ/mol).

You simply hydrolyze them

Phosphoric acid anhydrides

Electrostatic repulsion

Stabilization of products by ionization and resonance

These are energetically equivalent to ATP.

degradative pathways are stimulated.

degradative pathways are inhibited.

Redox (shorthand for oxidation-reduction) reactions describe all chemical reactions in which atoms have their oxidation number (oxidation state) changed.

• Oxidation is the loss of electrons
• Reduction is the gain of electrons

Flavins and Nicotinamides

H+ and two e^-

• Flavin Adenine Mononucleotide
• FMN (oxidized form); FMNH2 (reduced form)

• Flavin Adenine Dinucleotide
• FAD (oxidized form); FADH2 (reduced form)
• involved mostly in the redox of C=C bonds

• Nicotinamide Adenine Dinucleotide
• NAD+ (oxidized form); NADH (reduced form)

• Nicotinamide Adenine Dinucleotide Phosphate
• NADP+ (oxidized form); NADPH (reduced form)involved mostly in the redox of C=O bonds

The region of the enzyme that binds and acts upon the substrate

Binding = few or many weak attractions that can be reversed like, ion to ion, H-bond, van der Waals

are enzymes that change their conformation upon binding of an effector

Generally crevice or cavern on surface of enzyme

Active site has a rigid shape that is complementary to that of the substrate

The molecule is less stable, therfore higher energy

The shape of the enzyme adjusts to fit the proper substrate upon binding of the substrate

It is an allosteric enzyme

It will increase the rate only to a point, then it flattens out

The temp will increase the rate until the heat is so excesssive that it denatures the enzyme

The concentration of substrate at which 1/2 of the active sites are filled

The substrate can be eliminated from the denominator of the equation

Below Km

Never, the enzymes are never completely used

It is the direct measurement of the catylic product under saturated substrate conditions (turnover number). The maximum number of substrate molecules converted to product by the enzyme molecule per unit of time

Generally molecules which resemble or mimic a particular enzymes substrate(s).

The destruction or modification of an essential amino acid required for enzyme activity. Frequently, this is due to some type of covalent link between enzyme and inhibitor.

Inhibits acetocholinesterace

Inhibits the enzymes that help make bacterial cell walls

Inhibits enzymes with active site Serine, Acetocholinesterace (degrades acetylcholine)

Used to treat gout, it inhibits the reaction at two places

Swamp out substrate binding, and product formation is suppressed

Likewise large amounts of substrate swamp out inhibitor binding.

Ethanol competes for the same enzyme. Therefore administration of ethanol occupies the enzyme thereby delaying methanol metabolism long enough for clearance through the kidneys.

To characterize and compare the effectiveness of inhibitors relative to Km.

The tighter the binding, and hence the more effective an inhibitor is.

The enzyme does not bind the substrate as well in the presence of inhibitor and therefore “Km” increases

• Inhibitor and substrate DO NOT have common shape

• Binding is NOT at active site but causes a change in the substrate binding site

• Large amounts of inhibitor prevent access of substrate to binding site

• Large amounts of substrate CANNOT overcome inhibitory effect

Whether there is a substrate or not

Allosteric inhibitors bind on a different chain then the active site

• Allosteric enzymes are enzymes that change their conformation upon binding of an effector.
• Do not follow Michaelis-Menten Kinetics

A molecule separate from the substrate, activating and inhibiting effectors that bind at allosteric sites

Substrates acting as effectors are said to be homotropic effectors.

Substrate itself induces distant allosteric effects when it binds to the catalytic site

1. Bond Strain: Strains substrate bonds, which facilitates attaining the transition state.

2. Proximity and Orientation: Binding brings molecules into proximity and helps to properly orient reactive groups.

3. Acid/Base Catalysis: Required catalytic proton donors (acids) or acceptors (bases) are supplied by catalyst.

4. Covalent Catalysis: The reaction is facilitated by formation of a covalent intermediate between the enzyme (or coenzyme) and the substrate.

A serine, a histidine, and aspartic acid, regardless of specificity of cleavage.

Peptidoglycan hydrolysis, part of ‘innate’ immunity (breaks down cell walls of bacteria). Lysozyme is found in tears, saliva, nasal secretions and lysosomes.

Digestive enzyme, activated by proteolysis

Large bulky amino acids like Phenylalanine

Both acid and base qualities, where it can either donate or recieve a proton

Three amino acid residues found inside the active site of certain protease enzymes: serine (S), aspartate (D), and histidine (H). They work together to break peptide bonds on polypeptides.

The hydrolysis of the serine proteases like Chymotrypsin

Metal ions

a. They bind substrates to orient them for catalysis

b. Through redox reactions gain or loss of electrons.

c. Electrostatic stabilization or negative charge shielding

Carbonic Anhydrase, because the K_cat/K_m is very high

In the lungs and capillaries respectively

The statins have Ki values several orders of magnitude lower than the Km for the substrate

a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity.

coenzymes

Prosthetic groups

Contain multiple protein subunits

The amount of available enzyme