Comp 3 P1 Fall

Enzyme

is a biological catalyst that speeds a biochemical reaction by lowering the activation energy of the chemical reaction

Enzymes are

• protein in nature

• decrease activation energy thus speeding up reaction rate

Activation energy

the minimum amount of energy that is required to activate the reactants to a condition in which they can convert to products

Reaction rate

the speed at which a chemical reaction proceeds

Basic characteristics of enzymes

• high specificity

• active site binding

• catalytic efficiency

• regulation

• location

Enzymes are NOT consumed in the chemical reaction

true

Specificity

enzymes can distinguish and bind to a specific substrate from multiple compounds similar in structure

Active site binding

The three-dimensional arrangement of the binding site of the enzyme is composed of amino acid chains that allow the reacting portions of the substrate to approach and bind to the enzyme from the appropriate angles

Catalytic efficiency

Reactions that are enzyme-catalyzed reactions are very efficient (10^3 – 10^8 faster
than uncatalyzed reactions)

Regulation

The enzymatic activity is regulated by different molecules/pathways in order to respond to the cellular needs of product formation. These molecules can be
activated or inhibited by external drugs (serve as a drug target

Location

Enzymes are localized within the cells to isolate the reaction substrate or product from other competing reactions and organize the enzyme activity/pathways
within the cell

Apoenzyme

the inactive enzyme form without nonprotein moiety (enzyme without cofactor)

Holoenzyme

the active form of the enzyme with its nonprotein component (enzyme with the cofactor)

Cofactor

nonprotein moiety that is a metal ion in nature (e.g., Zn+2 or Fe+2)

Coenzyme

nonprotein moiety that is organic in nature (can either be cosubstrate or prosthetic group)

Cosubstrate

a small organic molecule that is transiently associated with the enzyme (dissociate from the enzyme in an altered state)

Prosthetic group

a small organic molecule that is permanently associated with the enzyme

In order for enzymes to carry out their catalytic activities, two strategies are employed:

1. Depend on the amino acid residues within the active site to provide proximity and orientation and directly bind with the substrate (all enzymes stabilize the transition state via electrostatic interactions but not all enzymes form covalent intermediates)

2. Depend on cofactors or coenzymes to provide a functional group with the right size, shape and properties to participate in the catalytic process of the enzyme

Cofactors

• metal ions in nature

• help binding to substrate OR stabilize developing anions in the reaction due to their positive charges (act as electrophiles)

Co-enzymes are usually synthesized from

vitamins

Activation-transfer coenzymes

Participates in the catalysis reaction by directly binding to the substrate with a covalent bond

Features of Activation-transfer coenzymes

• have a specific chemical group that binds to the enzyme

• have a separate and different functional group that participates in the catalytic reaction by binding covalently and directly to the substrate

• depend on the enzyme for additional catalytic power specificity for the substrate (coenzymes are NOT functional without enzymes)

coenzymes are NOT functional without enzymes

true

Oxidation-reduction coenzymes:

This type of coenzymes is involved in
oxidation-reduction reactions catalyzed by oxidoreductases

• do not form covalent bonds

Effect of temperature on enzyme activity

• The reaction rate increases with increasing the temperature until maximum velocity is reached

• Once the peak (maximum) velocity is reached
  at a certain temperature, the reaction rate starts to decrease due to denaturation of the enzyme as a result of high heat.

Effect of pH on enzyme activity

The effect of pH on enzymatic activity is enzyme specific. Some enzymes and substrates need to be in the ionized or non-ionized from in order to react

• extreme pH can lead to denaturation

Describes the induced-fit binding of enzymes and
substrates

The binding of the substrate to the enzyme prompts a conformational change of the enzyme that enhances substrate-enzyme binding

Velocity is the rate of

formation of product per unit time

Factors affecting the velocity of enzymatic reaction are

termp, pH, and substrate concentration

Vmax

Maximum velocity of the reaction

Km

Michaelis constant

[S]

concentration of the substrate

Vi =

(Vmax * S) / (Km + S)

Significance of Michaelis-Menten Equation:

provide a quantitative way of
describing the dependence of rate of enzyme catalyzed reactions on substrate
concentration by relating the initial velocity (vi) to the concentration of substrate [S] and the two parameters Km and Vmax

High Km means

low affinity

Low Km means

high affinity

Effect of substrate concentration on enzymatic reaction

The rate of enzyme-catalyzed reaction
increases as the concentration of the substrate
increases (proportional relationship) until
maximum velocity (Vmax) is reached

If a reaction does NOT follow MM kinetics the plot will be

sigmoidal

The higher the Km value,

the higher the amount substrate needed to reach the same half maximum velocity

The higher the Km value,

the lower the affinity of the enzyme to the substrate

Kcat (turnover number) is the number of

molecules of substrate converted to
product per enzyme molecule per unit time

The higher the turnover number (Kcat)

the higher the number of molecules produced
per unit time (the higher the efficiency of the enzyme)

Competitive inhibitors:

• structurally similar to the substrate

• both inhibitor and substrate compete for the active site

• Km increases (binding affinity decreases), no effect on Vmax

Non-competitive inhibitors

• The inhibitor binds to the allosteric site of the enzyme.

• No effect on KM,
  Vmax decreases

Uncompetitive inhibitors

• inhibitor binds to allosteric site AFTER the formation of enzyme substrate complex

• Both Km and Vmax decrease

Suicide inhibitors

• inhibitor binds to active site of enzyme with strong covalent bonds

• irreversible reaction

• No effect on Km

• Vmax decreases

The main goal of enzymes regulation is to

control processes to respond to changes in the cellular environments and overall to match body’s requirement (by up reg or down reg)

An enzyme pathway:

a series a sequential reactions in which the product of one reaction is the substrate for the next one (usually there is a different enzyme for each step)

Committed step

the first step in the pathway that initiates the unique pathway

Rate limiting step

• slowest step of the rate reaction

• irreversible

Regulatory enzyme is the enzymes

catalyzing the rate limiting step in the pathway

Regulation through conformational change

• In most of the rate-limiting enzymes, regulation of enzymatic activity occurs via regulatory mechanisms that induce conformational changes of the enzyme

• affects the active site

Mechanisms that induce enzymatic conformational changes

• allosteric enzyme regulation

• covalent modification

• protein protein interactions

• proteolytic cleavage

Allosteric enzymes regulation

• catalyze the committed step

• regulated by allosteric modifiers

• bind to the allosteric site of the enzyme (non covalently)

• contain 2+ subunits and exhibit a positive cooperativity

Allosteric activators

• binds to allosteric site of the enzyme in the R state

• The binding induces a conformational change of the active site

• increases the affinity of the enzymes to the substrate

Allosteric inhibitors

• binds to the allosteric site of the enzyme in the T states

• The binding induces a conformational change of the active site

• Decreases the affinity of the enzyme to the substrate

The addition of allosteric activator will

decrease Km

No effect on Vmax

Allosteric inhibitors can

increase Km alone OR

increases Km and decrease Vmax

Covalent modification (phosphorylation and dephosphorylation)

• regulation via the addition or deletion of a phosphate group

• phosphorylation catalyzed by protein kinases

• Dephosphorylation by protein phosphatase

Other groups that can be added via covalent modification include

acetyl, ADP ribose, lipid moieties

Proteins protein interactions

• regulation by direct interaction with other proteins in the cell

• conformational change leads to activation or inhibition

• Example: G protein

Monomeric G protein

1. Where G protein bind to GTP their conformation change allows them to bind to the target protein

2. G Protein hydrolyzes its bond with GTP to GDP and phosphate → target protein dissociates

3. Bound GDP in the inactive G protein will be replaced with GTP

Proteolytic cleavage

• undergo cleavage process during synthesis while others are stored or secreted as proenzymes

• INACTIVE WHEN SYNTHESIZED

Why are proenzymes synthesized as inactive precursors?

To prevent the cleavage of the targeted proteins prematurely at their sites of synthesis or secretion

Proenzymes (zymogens)

precursor (parent) proteins that must undergo proteolytic cleavage to be activated (becomes fully functional)

Regulation of enzyme synthesis

• induction (increases synthesis) or repression (decrease) is a slow process

• regulated by rate of gene transcription

Induced-fit Model

• views enzymes as adaptive and flexible rather than ridged

• the binding of the substrate to the
  enzyme “induces” conformational changes of the enzyme, allowing it to rearrange the critical functional groups needed to the formation of the E-S complex

Although we write/draw amino acids as neutral molecules, the

actual structures are ionic and depends on the pH of the medium

Scissile bond

the bond in the substrate that is subject to enzymatic cleavage

Mechanisms of enzyme catalysis

• proximity and orientation

• acid base

• covalent

• metal ion

• cofactor

Proximity and orientation catalysis

the enzyme force substrates to bind in a manner that places reactive groups in the appropriate orientation

Acid Base Catalysis

functional group on the protein either donates a proton (acid catalysis) or accepts a proton (base catalysis)

Covalent catalysis

substrate is covalently linked during the course of the reaction to an amino acid side chain at the active site of the enzyme

Metal ion catalysis

Many enzymes contain required metal ions to allow catalysis to occur

Cofactor

specific cofactor for an enzyme forms a covalent bond with the substrate during the course of the reaction.

Chymotrypsin

digestive enzyme that catalyzes the hydrolysis of peptide bonds in denatured proteins

• belongs to serine protease family

hydrolysis

the use of water to lyse (break a bond)

Proteolysis

the hydrolysis of a peptide bond in a protein

In the absence of chymotrypsin

• the hydroxyl group from
  water (negatively charged) attacks the carbonyl carbon atom
  (the is partially positively charged).
  - This leads to the formation of an unstable tetrahedral
  oxyanion intermediate (the transition state complex)
  - The electrons return from the oxygen atom back to the
  carbonyl carbon and the nitrogen atom leaves.
  - The remaining proton (H+) binds to the electron rich
  nitrogen atom to form an amino group.

What happens in the presence of chymotrypsin?

In the presence of chymotrypsin, the oxyanion intermediate is formed using the electrons
from the hydroxyl group of the serine residue of the chymotrypsin

Chymotrypsin catalysis steps

1. proximity and orientation

2. nucleophilic catalysis

3. acid base

4. stabilization

5. covalent

What happens to energy levels during
chymotrypsin-catalyzed reaction?

Decreases until the complex is in its most unstable state, then it increases and forms products

Metal ion catalysis

• have a positive charge

• 3 mechanisms:

  • assist in binding of substrate

  • stabilize the developing anions during rxn

  • accept and donate electrons during oxidation reduction rxns

HIV protease

• The cleavage of polyprotein precursor to individual mature proteins in performed by HIV protease enzyme

• belongs to aspartic proteases class

HIV-1 protease mechanism of action

acid base catalysis

What are CYP450 isozymes?

• heme-thiolate proteins that are synthesized in the liver

• considered monooxygenase enzymes

• found in other locations as well

Isozymes

• Isozymes (isoenzymes) are a group of enzymes that catalyze the same reaction but have different structures

• All living systems require multiple molecular forms of certain enzymes to maximize biological capacity

CYP450 structure

The highest structural conservation is found in the
core of the protein around the heme, hence, the
common mechanism of electron transfer and
oxygen activation is similar between different
CYP450 enzymes

Heme structure in CYP450 enzymes

type-b hemoproteins

Functions of CYP 450 enzymes

The function of CYP450 enzymes is organ-specific (depends on the organ where the specific enzyme is expressed)

The breakdown or alteration of drugs in phase 1

is catalyzed by CYP450

Phase 1 of drug metabolism

the drug is altered or broken down into intermediate metabolites

Phase 2 of drug metabolism

the intermediate metabolites are conjugated to
become more soluble and then expelled from the body in the urine

R-H + O2 + NADPH + H →

R-OH + H2O + NADP+

Factors affecting CYP450 enzymes expression and function

• CYP450 induction

• CYP450 inhibition

• Polymorphism

• Environmental factors

CYP450 inducers increase CYP450 enzyme activity by increasing enzyme synthesis, leading to

the result of CYP450 enzyme induction is acceleration of drug metabolism by the targeted
enzyme

CYP450 inhibitors decrease CYP450 enzymatic activity by inhibiting enzyme synthesis

The result of CYP450 enzyme inhibition is the reduction of drug metabolism by the targeted
drug

Polymorphism

the hereditary genetic variability among humans or certain human populations in drug metabolizing enzyme genes

Loss-of-function polymorphisms

Lead to poor metabolizers; people who inherit inactive genes that leads to deficient enzymatic activity

Gain-to-function polymorphisms

Lead to ultra-extensive metabolizers; people who have amplified gene expression and have enhance
enzymatic activity