14.1 Regulation of Respiration

True

Metabolic pathways are precisely controlled and regulated at the level of individual enzymes. True or False?

Metabolic Flux

The rate at which metabolites (reactants and products) move through a pathway. It is affected by many things. Shifting flux may involve different concentrations of metabolites, regulatory steps, or alternative pathways (hormonal signaling or other metabolic pathways).

Types of Enzymatic Regulation

• Effect

• Mechanism

• Connection to Pathway

Any combination of effect – mechanism – connection is possible, but does not have to be all three types simultaneously.

Types of Effects on Enzymes

Inhibition (repression) and activation.

Types of Enzyme Mechanisms

Regulator binding in active site, regulator binding an allosteric site, or covalent modification.

Allosteric Regulation

Occurs when a regulatory molecule binds to a protein at a site distinct from the active site (the allosteric site), causing a shape change that turns the protein's activity up or down. May affect substrate binding, product release, and/or rate of catalysis.

Cooperativity

When the substrate itself can serve as an allosteric activator. It binds to one active site so that the activity of the other active sites goes up. This is considered allosteric regulation because the substrate affects active sites far from its binding site.

Covalent Modification

The formation or breaking of covalent bonds via the addition or removal of chemical groups. Can be reversible, though some forms are irreversible. Alters the primary and tertiary structure of the enzyme itself.

Ex: phosphorylation

Types of Connections to the Metabolic Pathway

• Feedback regulation

• Feed-forward regulation

• General regulation

Feedback Regulation

When the downstream product is a regulator.

Feed-Forward Regulation

When the upstream reactant is a regulator.

General Regulation

No connection to the metabolic pathway. Ex: cofactors (non-protein chemical compound or metal ion required by an enzyme to catalyze a reaction)/coenzymes (coenzyme is a specific subtype of cofactor that is an organic, carbon-based molecule)

Compartmentalization

Enables the segregation of enzymes (often in an organelle) involved in different metabolic pathways, regulating cellular processes.

Ex: digestive enzymes of the lysosome work best at a pH around 5, which is found in the acidic interior of the lysosome (but not in the cytosol, which has a pH of about 7.2).

Regulation of the Irreversible Steps

The three enzymes that have the biggest affect on flux in glycolysis are those that catalyze the three irreversible steps. These enzymes are therefore heavily regulated.

• Hexokinase

• Phospho-fructokinase 1

• Pyruvate kinase

By studying just what happens to these three enzymes, we get examples of almost every kind of regulation mentioned on the previous slide!

Glucose-6-Phosphate Crossroads

G6P can be used in:

• Glucose production (monosaccharide circulating in blood is the body's immediate + primary source of energy).

• Glycogen production (polysaccharide made of thousands of glucose molecules linked together and stored in liver/muscles for later use).

• Glycolysis (metabolic process in the cell cytoplasm that converts glucose to G6P by hexokinase).

• Pentose phosphate pathway (makes ribose for use in nucleic acids).

Product Inhibition

Where the product of an enzyme-catalyzed reaction reduces or slows down the activity of the enzyme that produced it.

Regulation of Hexokinase

Hexokinase is regulated through inhibition by G6P (product inhibition).

Product inhibition by G6P is a mix of competitive inhibition and allosteric inhibition, since G6P directly competes with the ATP binding site at the active site on the C-terminus and also binds to an allosteric site on the N-terminus.

Committed Step to Glycolysis

Making Fructose,1-6 bisphosphate (FBP) from F6P using PFK-1. FBP takes a lot of energy to make and is not involved in any other major metabolic pathway.

Phosphofructokinase-1 (PFK-1)

The enzyme involved in the this step is committed step to glycolysis (F6P to FBP) and is the main rate-limiting enzyme. It shifts between an active state and an inactive state. F6B substrate only binds to the active site in the active state.

Allosteric activators cause a shift to the active state, and inhibitors (ATP and citrate) would cause a shift to the inactive state.

This is typical for an allosteric enzyme. At low [substrate], there is a mix of inactive and active states, but as substrate concentration increases, more of the enzyme shifts to the active state.

Why does the enzyme kinetics curve have a sigmoidal shape that does not follow the classic Michaelis-Menten kinetics graph shape?

Adenosine Monophosphate (AMP)

PFK-1 allosteric activator. When a cell rapidly consumes ATP, it is broken down into ADP and AMP. Because the enzyme adenylate kinase converts two ADP molecules into one ATP and one AMP a small drop in ATP causes a large spike in AMP concentration. AMP functions as an early-warning signal that energy levels are low and that glycolysis is needed.

ATP and Citrate

PFK-1 allosteric inhibitors. Citrate is the first product made in the mitochondrial citric acid cycle, so high levels of citrate mean the downstream energy-producing machinery is backed up.

First F6P will buildup, followed by G6P. F6P will form more G6P since the isomerase reaction is reversible. G6P inhibits hexokinase (glucose to G6P) so more glucose will accumulate. More glycogen, glucose, and ribose will be made and glycolysis will decrease.

If PFK-1 is inhibited, what may happen?

The Last (10th) Step in Glycolysis

The 3rd irreversible step in glycolysis is conversion of phosphoenol pyruvate to pyruvate by pyruvate kinase (PK).

Transamination

The transfer of an amino group onto an amino acid from another amino acid. How alanine is made directly from pyruvate.

Fructose 1,6 bisphosphate (FBP) for PK Regulation

A positive allosteric regulator of PK. An example of feed-forward activation, where an earlier product in the pathway activates a later one (opposite of feedback inhibition).

PK Covalent Modification Activator

PK can also be phosphorylated directly (covalent modification). Triggered due to a signaling pathway activated by the hormone glucagon, which is activated by low blood glucose levels.

PK Feedback Inhibition

Caused by ATP accumulation (a result of later steps in glycolysis) and the amino acid alanine (made directly from pyruvate, an earlier step in glycolysis).

Gluconeogenesis

Gluconeogenesis is when glucose is made from pyruvate, but is is not a direct reverse of glycolysis because of the last irreversible step made during glycolysis. Gluconeogenesis needs to create an intermediate in order to go in the reverse direction of glycolysis.

Glycogenolysis

A type of glycogen metabolism regulation. The biochemical breakdown of stored glycogen into glucose units. Glucose-1-phosphate (G1P) is made, which gets converted to G6P.

G6P is an allosteric inhibitor of glycogen phosphorylase. Has opposing allosteric effects on glycogen breakdown and synthesis, which helps the cell avoid wasting energy.

Glycogenesis

A type of glycogen metabolism regulation. The building of glycogen from glucose. It starts when G6P is converted to glucose-1-phosphate (G1P).

G6P is an allosteric activator of glycogen synthase (catalyzes the addition of an glucose molecule to the end of a growing glycogen chain). Has opposing allosteric effects on glycogen breakdown and synthesis, which helps the cell avoid wasting energy.

Triglycerides

The structures that fat is stored as.

Fatty Acid Activation

The preparatory step required before a fatty acid can be broken down for energy during beta-oxidation.

ATP is used to break up the triglyceride and charge each fatty acid with CoA to make fatty acyl-CoA. Since ATP → AMP + PP (uses 2 high-energy bonds), this counts as two “ATP equivalents” used per fatty acid to charge it with CoA.

Beta-Oxidation of Lipids

A 2-carbon acetyl-CoA is broken off the end of the fatty acid chain. Every cycle of beta oxidation produces:

• 1 NADH

• 1 FADH2

• 1 Acetyl-CoA (used in same way as if it was from glucose)

This process continues until the entire fatty acid is broken down. An 18-carbon fatty acid will undergo 8 rounds of beta-oxidation (#C/2 - 1). Each round generates one NADH and one FADH2 split into 9 acetyl-CoA (#C/2).

Lipogenesis

Lipid synthesis. Acetyl-CoA accumulation leads to excess citrate. When citrate is in excess, it shifts metabolism back to building Acetyl-CoA. Acetyl-CoA build-up leads to both this and cause accumulation of ketone bodies (ketosis).

This is the desired outcome of the keto diet, when fat is burned instead of carbohydrates. Can be dangerous if ketone bodies build up too much, leading to ketoacidosis.

Deamination

When amino acids 1st have their amino group removed.

The Mandatory Preparatory Steps Amino Acids for Energy

Amino Acids 1st have their amino group removed (deamination) or transferred to another amino acid to build (transamination).

The remaining carbon skeleton of the amino acid enters the respiration pathway one of the following ways:

• converted to Acetyl-CoA

• converted to pyruvate

• converted to other molecules within the citric acid cycle

While amino acids can be used for fuel, they generally are not. Only if amino acids are in excess are they used for food.

Eukaryotic Metabolism

All food energy metabolism pathways are interwoven. Cellular “decision making” in food metabolism depends on:

• Concentration of intermediates and products (including ATP), some of which may regulate other steps

• Extracellular regulation (hormones)

All organisms need to make these decisions regarding how to use energy, including non-eukaryotes.

Rate-Limiting Enzymes

Glycolysis, hexokinase, phosphofructokinase-1, and pyruvate kinase are considered these due to their regulation. PFK-1 is the main rate-limiting enzyme in glycolysis – remember after this step the cell is “committed”.