Enzyme Regulation

Question 1

Allosteric Regulation

Answer 1

• Importance of regulation in metabolic pathways
• Allostery
• ATCase and glycogen phosphorylase as example of allosteric regulation
• New approaches to allostery

Question 2

Metabolic Pathways

Answer 2

• Life requires a complex set of chemical reactions
• Many metabolic pathways use the same starting materials
• Interconnected in many ways
• Regulation of the flux through these pathways is key to maintaining organisms health

Question 3

Means to Regulate Metabolic Flux

Answer 3

• Control the amount of enzyme
• Transcriptional or translational control
• Control the activity of the enzymes present

Question 4

Ways to modulate enzyme activity

Answer 4

• Allosteric Enzymes: modify activity with other molecules that enhance or inhibit activity
• Phosphorylation: Covalent Modification
• Zymogens: Convert inactive to active forms
• Tight Binding Proteins: Protein-Protein interactions to regulate enzyme activity

Question 5

Allosteric Enzymes

Answer 5

• Usually larger symmetric multi-subunit enzymes
• Allosteric enzyme activities are regulated by small metabolites, which are substrates, activators, and inhibitors that bind to sites other than the active site
• Exists in 2 forms, active R state and less active T state
• Frequently exhibit cooperative binding of substrate to the active site
• Kinetics of the enzyme show a sigmoidal response to increased substrate concentration

Question 6

Models of Allosteric Interactions - MWC Model

Answer 6

• Symmetry Model
• Each subunit exists in either the T or R state
• Ligand binds to either state
• Molecular symmetry of the oligomer is maintained

Question 7

Models of Allosteric Interactions - KNF Model

Answer 7

• Sequential model, induced fit
• Ligand binding to one subunit changes the affinity for adjacent subunits
• As more ligands are bound, more subunits adopt high affinity conformation

Question 8

Effector Molecules - Homotropic Effector

Answer 8

Substrate binds one subunit and increases affinity at another

Question 9

Effector Molecules - Heterotropic Effector

Answer 9

Binds at sites other than the active site and are not substrate

Question 10

Effects of Activators

Answer 10

• Stabilize the R state of the enzyme
• Activity is increased by increasing Vmax or decreasing Km
• Also tend to decrease cooperative binding of substrate

Question 11

Effects of Inhibitors

Answer 11

• Inhibitors stabilize the T state of the enzyme
• Activity is decreased by decreasing Vmax or increasing Km
• Also increase cooperative binding of substrate

Question 12

Asparate Transcarbamoylase

Answer 12

• A model allosteric enzyme that catalyzes the first commited step in pyrimidine biosynthesis
• Catalyzes the initial step in the synthesis of pyrimidines, Cytidine, Thymidine, and Uridine

Question 13

Asparate Transcarbamoylase Mechanism

Answer 13

• Ordered binding of substrates
• Carbonyl Phosphate binds first
• Binding of CP causes changes in the local structure and creates the ASP binding site
• ASP binding cause both tertiary and quaternary structural changes

Question 14

Cooperative Binding of Substrate

Answer 14

• The binding of aspartate is cooperative
• ATP is a + effector
• CTP is an inhibitor
• CTP and UTP are required for complete inhibition

Question 15

Why are ATP, UTP, and CTP regulators of ATCase

Answer 15

• CTP and UTP are products of the pyrimidine pathway “feed back inhibition”
• ATP is a purine, which must be kept in balance for DNA and RNA synthesis

Question 16

Structural Studies of the Synergy of CTP, UTP and Mg2+

Answer 16

Kinetic Experiments and new structures determined with careful controlled Mg2+ concentrations show that Mg2+ cations and UTP are required for full allosteric inhibition of ATCase

Question 17

ATCase

Answer 17

• An allosteric enzyme regulated by feedback inhibition and feed forward activation to maintain the balance of the pyrimidine purine pools in diving cells
• The structural changes upon binding of substrate and effectors support the MWC model of allostery

Question 18

Glycogen Phosphorylase

Answer 18

• Catalyzes the stepwise removal of glucose residues from the glucose storage molecule glycogen

Question 19

Summary of Traditional Approach to Allostery

Answer 19

• Allosteric enzymes regulate the majority of metabolic pathways in the cell
• Homotropic allosteric effects lead to cooperative binding of substrate, allowing greater sensitivity to changes in substrate concentrations
• Heterotropic effectors, metabolites in the pathway, enhance or decrease enzyme activity in response to changes in the cellular requirements for the products of the pathway

Question 20

Models of Allosteric Interactions

Answer 20

• Entropy driven, dynamic model
• No structural changes between active and inactive form
• Effector changes side chain or backbone dynamics of the protein

Question 21

Changes in Protein Dynamics

Answer 21

• Dihydrodipicolinate synthase catalyzes the first committed step in the biosynthesis of lysine
• The tetrameric enzyme is allosterically inhibited by lysine
• No change in structure, just mobility

Question 22

Ensemble Model of Allostery

Answer 22

• Protein exists as an ensemble of structures with different domains folded
• Effector binding changes the distribution of forms, increasing or decreasing the activity

Question 23

Morpheein Model of Allostery

Answer 23

• Subunits arrange in different quaternary structures by dissociation and reassociation into a different oligomer
• One form is active and the other is not
• Porphobilinogen synthase

Question 24

Medicinal Chemistry and Allostery

Answer 24

• Enzyme active sites are more conserved than allosteric sites
• Allosteric drugs can be more species specific

Question 25

Zymogens: Enzymatic Control by Proteolysis

Answer 25

- Some enzymes are made in an inactive form - Activation requires cleavage of peptide bonds - Chymotrypsin requires hydrolysis at three sites for complete activation - Zymogens of intestinal proteases are made in the pancreas, activated in the small intestine

Question 26

Summary - Allosteric Effectors

Answer 26

Metabolites that regulate enzyme activity by binding to sites different than the active, both positive and negative effectors

Question 27

Summary - Zymogens

Answer 27

- Proteolytic enzymes are synthesized as inactive enzyme - Cleavage activates the enzyme in the appropriate location

Question 27

Summary - Covalent Modification

Answer 27

- Addition of small groups to enzymes, phosphorylation - The modification can enhance or decrease enzyme activity

Question 28

Summary - Tight Binding Protein Inhibitors

Answer 28

BPTI binds to trypsin so tightly that catalysis cannot occur