Lecture 4: Enzyme Regulation Part 1

Oxidoreductases (Hint: 2)

Oxidation-reduction reaction

Example: Lactate dehydrogenase

Dehydrogenase (Hint: 3)

Catalyzes dehydrogenation

Remove H/e^-

Oxidation reaction

Transferases (Hint: 2)

Group transfer

Example: Nucleoside monophosphate kinase (NMP kinase)

Kinase

Transfers phosphate groups

Hydrolases (Hint: 2)

Catalyze hydrolysis reactions (transfer of function groups to water)

Example: Chymotrypsin, Amylase and Lipase

Amylase

Hydrolyzes starch

Lipase

Hydrolyzes lipids

Trypsin

Breaks down proteins (protease)

Lyases (Hint: 2)

Addition or removal of groups to form double bonds or ring structures

Example: Fumarase and Cellulose synthase

Fumarase (Hint: 2)

Used in basic metabolism

Creates double bonds

Cellulose Synthase (Hint: 2)

Synthesis of cellulose (polymer of glucose)

Forms ring structures

Isomerases (Hint: 2)

Isomerization (intramolecular group transfer)

Example: Triose Phosphate Isomerase

Isomer

Same formula, different spatial arrangement

Ligases (Hint: 4)

Ligation of two substrates at the expense of ATP Hydrolysis

Puts Okazaki fragments together in DNA replication

Make bonds to seal them together -> covalent bonds -> Uses energy from ATP

Example: Aminoacyl-tRNA Synthetase

Aminoacyl-tRNA Synthetase (Hint: 4)

t-RNA

Transfers amino acid to ribosome to make protein

Puts amino acid onto tRNA

Uses ATP to connect (no ATP = synthase)

Allosteric Control

Regulation of a protein by binding an effector molecule at a site other than the enzyme’s active site

Allosteric

Elsewhere in space

Allosteric Enzymes

Multi-subunit enzymes

Aspartate Transcarbamoylase (ATCase)

Catalyzes the first step in pyrimidine synthesis

Sigmoidal Curve (Hint: 4)

Results from cooperativity between subunits/substrate-binding sites

Most allosteric enzymes have this type of kinetics

S-shaped “sigma”

Lag phase in the beginning

Homotropic Effects (Hint; 2)

The effects of substrates on allosteric enzymes

Similar movement effects

Concerted (“all or none”) mechanism for allosteric enzymes (Hint: 2)

All active sites are in the same state, either T or R

In one enzyme, if 1 picks up substrate, all pick up substrate

Cooperativity

All subunits need to work together

Catalytic Subunit (Hint: 2)

Catalyze reaction

Bind to substrate

Regulatory Subunit (Hint: 2)

Control enzyme activity

Allosterically binds

Bi-Substrate Analog (Hint: 2)

Molecule that looks like 2 substrates

Come up with molecules that looks like reaction intermediate

T-State (Hint: 4)

Tensed

More compact

Less functional

Removal of substrate/addition of allosteric inhibitor make T-state

R-State (Hint: 3)

Relaxed

More functional

Substrate binding makes it relaxed

List the 6 major classes of enzymes

Oxidoreductases

Transferases

Hydrolases

Lyases

Isomerases

Ligases

Enzymatic Activity is Regulated in what 4 Principal Ways?

Allosteric control: Controlled by molecule that is binding somewhere other than active site

Multiple forms of enzymes: Use different versions of same enzyme

Reversible covalent modification: Phosphorylation and acetylation; Add covalent bond and change shape/activity

Proteolytic activation: Some need to be digested before it can work

Describe ATCase Reaction (Hint: 4)

Allosterically inhibited by the end product of its pathway, CTP, in an example of feedback inhibition

If high CTP, it binds to ATCase, changes shape, and stops enzyme

CTP exerts its effects by binding at a distinct regulatory or allosteric site on ATCase

Displays Sigmoidal Kinetics and does not display Michaelis Menton Kinetics

Describe the ATCase Active Site (Hint: 5)

Consists of 6 catalytic and 6 regulatory subunits (c₆r₆)

Behaves like one protein (enzyme)

Active sites are located at the interface of the catalytic subunits

Each catalytic subunit has its own active site

Identified by use of the bi-substrate analog PALA

Describe PALA

Binding of PALA (substrate) causes structural changes that convert the compact, less active T state into the expanded, active R state

Describe the T and R State in terms of ATCase (Hint: 4)

The T state has a low affinity for substrate and has low catalytic activity, while the R state is the most active form

The two states are in equilibrium, with the T state being favored in the absence of substrate and in the presence of CTP

Binding of substrate disrupts the equilibrium in favor of the R state, this involves cooperativity (when substrate starts binding to catalytic trimer, all 3 subunits are activated)

Binding of CTP to the regulatory sire of ATCase alters the T-to-R equilibrium in favor of the T state, decreasing net enzyme activity (more CTP = more T-state enzymes)

What mediates allosteric interactions in ATCase? (Hint: 2)

Large changes in Quaternary structure

If there is allosteric effect of ATCase (CTP), large scale changes of structure of enzyme

The sigmoidal kinetic curve of allosteric enzymes allows what?

Increased sensitivity to changes in substrate concentration

ATCase follows what mechanism?

Concerted (“all or none”) mechanism