Regulation of Enzyme Activity

• Many biological processes take place at a specific time; at a specific location and at a specific speed. • The catalytic capacity is the product of the enzyme concentration and their intrinsic catalytic efficiency. • The key step of this process is to regulate either the enzymatic activity or the enzyme quantity.

Reasons for regulation • Maintenance of an ordered state in a timely fashion and without wasting resources • Conservation of energy to consume just enough nutrients • Rapid adjustment in response to environmental changes

Regulation of Enzyme Activity 1.

Zymogen activation (Proteolytic cleavage of proenzyme)

2. Allosteric regulation 3. Association-disassociation 4. Covalent modification

Zymogen activation • Zymogens or proenzymes are inactive precursors of enzymes. • Activation involves the irreversible hydrolysis of one or more peptide bonds, resulting in an active form. • It is the conformational changes that either form an active site of the enzyme or expose the active site to the substrates. • A cascade reaction in general

Wide varieties • Hormones: proinsulin • Digestive proteins: trypsinogen, … • Funtional proteins: factors of blood clotting and clot dissolution • Connective tissue proteins: procollagen

Activation of chymotrypsinogen by proteolysis

Activation of trypsinogen by proteolysis

Proteolytic Enzymes of the Digestive Tract

Activation of Proinsulin to Insulin

Activation of Blood Clotting Factors • Clotting involves series of zymogen activations • Seven clotting factors are serine proteases involved in clotting cascade

X X X

X X

X

Digestive enzymes, blood clotting enzymes, and enzymes involved in bone and tissue remodeling catalyze reactions that would be disastrous if they occurred at inappropriate times or locations. For example, if proteolytic digestion of proteins occurred in the pancreas, they would start digesting the pancreas itself. Similarly, if blood clotting factors are activated when they aren’t needed, they will initiate blood clotting throughout the body. So, they are synthesized as inactive zymogens and are stored in this inactive state until they are needed.

Allosteric regulation • Allosteric enzymes are those whose activity can be adjusted by reversible, non-covalent binding of a specific modulator to the regulatory sites, specific sites on the surface of enzymes. • Allosteric enzymes are normally composed of multiple subunits (Catalytic and regulatory subunits) which can be either identical or different.

General Features of Allosteric Regulation: Effects • A positive effector activates the enzyme (an activator). • A negative effector inhibits the enzyme (an inhibitor). • Positive cooperativity increases substrate binding in an adjacent subunit. • Negative cooperativity decreases substrate binding in an adjacent subunit.

General Features of Allosteric Regulation

Kinetic plot of v versus [S] is sigmoidal shape

Feedback inhibition • An enzyme, early in the metabolic pathway, is inhibited by an endproduct. • Often takes place at the committed step of the pathway, the step which commits a metabolite to a pathway.

Covalent modification • A variety of chemical groups on enzymes could be modified in a reversible and covalent manner.

• Such modification can lead to the changes of the enzymatic activity.

Common modifications phosphorylation - dephosphorylation adenylation - deadenylation methylation - demethylation uridylation - deuridylation ribosylation - deribosylation acetylation - deacetylation

Covalent modification: Phosphorylation

Features of covalent modification • Two active forms (high and low) • Covalent modification • Energy needed

Features of covalent modification

• Amplification cascade (Active Csubunit)

-P -P

Features of covalent modification • Some enzymes can be controlled by allosteric and covalent modification

Favored

• Glycogen Phosphorylase

Favored

Top 4 reasons why phosphorylation is used to regulate enzyme activity 1. Phosphorylation is rapidly reversible, making it possible to quickly switch between active and inactive forms of an enzyme. 2. Phosphorylation is relatively inexpensive since it does not require the synthesis of new protein molecules. 3. Phosphorylation/dephosphorylation is rapid and its timing can be adjusted to meet the physiological needs of the cell. 4. Phosphorylation effects can be rapidly amplified via a kinase cascade.

Enzyme Regulation by Association/Disassociation • Acetyl-CoA Carboxylase ▫ acetyl-CoA + CO2 + ATP
malonyl-CoA + ADP + Pi ▫ 1St committed step in fatty acid biosynthesis ▫ A ctivated by citrate ▫ Inactivated by fatty acyl-CoA citrate

Unpolymerized (less active)

Fatty acyl-CoA

Polymerized (Active)

cAMP-dependent protein kinase

• Cyclic AMP-dependent protein kinase, also known as protein kinase A (PKA), is a 150- to 170-kD R2C2 tetramer in mammalian cells. • The two R (regulatory) subunits bind cAMP; cAMP binding releases the R subunits from the two C (catalytic) subunits.
C subunits are enzymatically active as monomers

Enzyme Regulation by Compartmentation organelle

Enzyme/metabolic pathway

Cytoplasm

Aminotransferases, peptidases, glycolysis, hexose monophosphate shunt, fatty acids synthesis, purine and pyrimidine catabolism

Mitochondria

Fatty acid oxidation, amino acid oxidation, Krebs cycle, urea synthesis, electron transport chain and oxidative phosphorylation

Nucleus

Biosynthesis of DNA and RNA

Endoplasmic reticulum

Protein biosynthesis, triacylglycerol and phospholipids synthesis, steroid synthesis and reduction, cytochrome P450, esterase

Lysosomes

Lysozyme, phosphatases, phospholipases, proteases, lipases, nucleases

Golgi apparatus

Glucose 6-phosphatase, 5’-nucleotidase, glucosyl- and galactosyltransferase

Peroxisomes

Calatase, urate oxidase, D-amino acid oxidase, long chain fatty acid oxidase

Regulation of E Quantity • The overall synthesis and degradation of a particular enzyme, also termed its turnover number, is one way of regulating the quantity of an enzyme. • The amount of an enzyme in a cell can be increased by increasing its rate of synthesis, decreasing the rate of its degradation, or both.

Regulation of E Quantity • Constitutive enzymes (house-keeping): enzymes whose concentration essentially remains constant over time • Adaptive enzymes: enzymes whose quantity fluctuate as body needs and well-regulated. • Regulation of enzyme quantity is accomplished through the control of the genes expression.

Controlling the synthesis • Inducer: substrates or structurally related compounds that can initiate the enzyme synthesis • Repressor: compounds that can curtail the synthesis of enzymes in an anabolic pathway in response to the excess of an metabolite

Controlling the degradation • Enzymes have a wide range of lifetime. LDH4 5-6 days, amylase 3-5 hours. • They degrade once not needed through proteolytic degradation. • The degradation speed can be influenced by the presence of ligands such as substrates, coenzymes, and metal ions, nutrients and hormones.

Degradation pathway 1- Lysosomic pathway: ▫ ▫ ▫ ▫

Under the acidic condition in lysosomes No ATP required Indiscriminative digestion Digesting the invading or long lifetime proteins

2- Non-lysosomic pathway (The UbiquitinProteasome Pathway): ▫ Digest the proteins of short lifetime ▫ Labeling by ubiquitin followed by hydrolysis ▫ ATP needed