Enzyme Catalysis Flashcards

Enzyme Catalysis

Enzyme Regulation Overview

Enzyme activity is regulated to be switched on (activated) or switched off (inhibited) through various mechanisms:

• Reversible binding of small molecules

• Alteration of enzymes by covalent modification

• Irreversible proteolytic activation

• Altering the total amount of an enzyme present

Reversible Binding

Competitive vs. Non-Competitive Inhibitors

Reversible inhibitors can be competitive or non-competitive, depending on whether they bind to the active site or a secondary site.

Catalytic Profiles

Competitive and non-competitive inhibitors result in different catalytic profiles.

• Competitive Inhibition: Increases the $K<em>m$ of the reaction, but the $V</em>{max}$ remains the same.

• Competitive inhibitors that bind at the active site are substrate analogs.

• This is not a common mechanism for controlling the activity of intracellular enzymes, but it is a common mechanism for drug action.

Allosteric Enzymes

Activation and inhibition of intracellular enzymes with allosteric properties is a general mechanism for controlling metabolism to meet the internal requirements of the cell.

• Many intracellular enzymes that are important control points in metabolism have allosteric properties.

• Allosteric enzymes have distinct regulatory and functional sites (i.e., quaternary).

• Exhibit cooperative activity (i.e., activity at one site influences activity at another), e.g., hemoglobin.

Mechanism

Allosteric enzymes oscillate between an active and an inactive conformation.

• Allosteric activators and inhibitors bind reversibly and lock the enzyme in the active or inactive conformation.

• Allosteric enzymes display a sigmoidal curve (“S” shape) when reaction velocity is plotted against varying substrate concentrations.

Control of Glucose Breakdown: Phosphofructokinase Example

The enzyme activity can be altered over a range of ~20 fold by the allosteric activator.

• Example: The enzyme Phosphofructokinase is a key regulator of glucose catabolism.

• F6P is the substrate.

• F2,6BP is the small molecule.

Reversible Covalent Modification

Alteration by covalent attachment of a modifying group.

• Commonly this is phosphorylation of hydroxyl (-OH) groups on amino acid side chains.

• This modification is reversible via dephosphorylation.

• This is like a molecule switch to turn activity “on” or “off”.

• Phosphorylation/dephosphorylation is not just common for regulating enzyme activity, but is as part of cellular responses to external signals, such as the binding of hormones or neurotransmitters to receptors and transporters.

• This allows control of metabolism or other cellular processes in response to external signals.

Kinases vs. Phosphatases

Proteins are phosphorylated by kinases and dephosphorylated by phosphatases.

• Phosphorylation occurs on Serine, Threonine, and Tyrosine.

• Phosphorylation adds two negative charges to a protein, which can alter its structure and function.

• Phosphorylation/dephosphorylation by protein kinases and phosphatases commonly occurs as part of cellular responses to external signals, such as the binding of hormones or neurotransmitters to receptors.

• It is one of the ways of changing the activity of an enzyme (or the function of other types of proteins) to control metabolism or other cellular processes in response to external signals.

Proteolytic Activation

Some proteins are inactive when synthesized and require activation by specific proteolytic cleavage.

• The inactive precursor is called a zymogen or proenzyme.

• Activation occurs once only in the life of the protein (irreversible).

• Examples:

  • Digestive enzymes

  • Blood clotting

  • Some hormones (e.g., insulin)

  • Some structural proteins (e.g., collagen)

  • Developmental processes

  • Apoptosis (programmed cell death) - caspases

Proteolytic Activation in Digestion

Proteolytic activation of digestive enzymes breaks down dietary protein to provide intake of amino acids.

• Context:

  • Stomach: Acidic environment and pepsin

  • Intestine: Proteolytic enzymes from the pancreas and proteases located in the plasma membranes of intestinal cells

• It is essential that gastric and pancreatic enzymes are not active until they:

  • Are in the correct location (not where they are synthesized)

  • Are in the environment where there are target proteins to digest

Zymogen Synthesis and Release

• The pancreas is one of the most active organs for synthesis and secretion of proteins in the body.

• Zymogens are synthesized in the acinar cells of the pancreas.

• Zymogen granules accumulate at the apex of the acinar cells in the pancreas.

• Hormonal signaling or nerve impulse causes their release into a duct leading to the duodenum.

Proteolytic Activation of Chymotrypsin

Trypsin cleaves chymotrypsinogen, with the two peptides connected by a disulfide bond.

• A $π$-chymotrypsinogen can react with another to form $α$-chymotrypsin, the stable form of the enzyme. It has 3 polypeptide chains linked by disulfide bonds.

• Activation changes the protein structure.

• The digestive proteins trypsin and elastase are homologs of chymotrypsin.

• Their overall structures are nearly identical, but they have very different substrate specificities.

Substrate Specificity

The different enzymes have different substrate specificity. They cleave the peptide backbone after amino acids with specific types of side chains:

• Chymotrypsin: hydrophobic side chains

• Trypsin: +vely charged side chains

• Elastase: small side chains

• The differences in substrate specificity are produced by changes in a few amino acids at the substrate-binding site.

• Concurrent activation occurs by trypsin activating all pancreatic zymogens.

• Trypsin itself is produced as a zymogen.

• The cells lining the duodenum secrete enteropeptidase, which cleaves and activates trypsinogen.

Pancreatic Trypsin Inhibitor (PTI)

The pancreas also produces a pancreatic trypsin inhibitor (PTI).

• Part of the PTI polypeptide chain is recognized as a substrate by trypsin and binds tightly at the active site.

• But the PTI is a very poor substrate and is only cleaved very slowly.

Why Synthesize PTI?

The PTI acts as an off-switch for any trypsin formed by accidental trypsinogen activation.

• Accidental activation of trypsin is particularly dangerous because of its position at the start of the proteolytic cascade of digestion enzymes.

• If trypsin is activated in the pancreas or the pancreatic ducts, it will lead to pancreatitis or tissue necrosis.

Digestion Summary

Following proteolytic activation in digestion:

• Digestive enzymes further degrade oligopeptides in the intestine.

• Amino acids, di- and tri-peptides are imported from the lumen into intestinal cells.

• Oligopeptides are first cleaved by intestinal aminopeptidase.

• Di- and tri-peptides are converted to amino acids by peptidases.

• Specific transporters import and export the amino acids.

• Amino acids are transported from the lumen to the bloodstream.

Begins in the stomach where the acidic environment denatures the proteins.

• Denatured proteins are digested further by pepsin, a protease that is maximally active at pH 2.

  • Continues in the lumen of the intestine using proteolytic enzymes from the pancreas.

  • Aminopeptidases in the plasma membrane of the intestinal cells complete the digestion.

Key Takeaways:

• Enzymes may be produced as zymogens, which require activation.

• Inhibitors may switch enzyme catalysis off.

Enzyme Regulation in Digestion: Lipases

$Triglyceride + H_2O \rightarrow Glycerol + 3 \text{ fatty acid chains}$

Triacylglycerides are converted to Monoacylglycerols by Pancreatic Lipase.

Lipase and Colipase

Colipase is secreted with the pancreatic lipase.

• It binds to the inactive lipase and helps to bind it to lipid micelles.

• This causes a conformational change in the lipase, exposing an active site in the region of the enzyme surface in contact with the substrate and “switching on” the enzyme.

Altering the Amount of an Enzyme Present

This can be controlled through the regulation of gene expression (DNA to RNA to protein) OR the degradation of existing cellular proteins.

Protein Degradation

Protein turnover occurs constantly.

• Proteins have varying half-lives, e.g.,

  • Crystallin - the life of the organism

  • Ornithine decarboxylase - 11 minutes

• Protein degradation regulates biological functions:

  • Gene transcription

  • Cell-cycle progression

  • Organ formation

  • Circadian rhythms

  • Inflammatory response

  • Tumor suppression

  • Cholesterol metabolism

  • Antigen processing

Ubiquitin

Ubiquitin (Ub) marks proteins for degradation, enabling protein turnover to be tightly regulated.

• Found in all eukaryotic cells.

• Highly conserved (yeast and human Ub differ by only 3 out of 76 residues).

• Attaches to target proteins with the assistance of other enzymes.

• The attachment of one Ub molecule is only a weak signal for protein degradation.

• Chains of four or more Ub molecules enhance the signal.

• Binds preferentially to a Lys in the target and to a Lys in another Ub.

• Determining factors for ubiquitin attachment to a target:

  • The N-terminal residue

  • PEST boxes

  • Cyclin destruction boxes

Errors in ubiquitin-mediated protein degradation are associated with early-onset Parkinson's disease and cervical cancer caused by HPV infection.

Proteasome

Ubiquitin places a target on the protein to be degraded.

• The proteasome degrades the ubiquitinated protein.

• The proteasome subunits split the protein into peptides and release ubiquitin for reuse.

• Peptides are then broken down into amino acids by other peptidases.

• The proteasome cap recognizes and unfolds ubiquitin-tagged proteins.

• The central portion of the proteasome catalyzes the target protein degradation.

• ATP hydrolysis provides the energy for this catalysis.