Enzyme Kinetics Flashcards
How are enzymes regulated?
- Proteolytic Cleavage (irreversible covalent modification)
- Reversible covalent modification
- Control proteins
- Allosteric inhibition
Proteolytic Cleavage
This is an irreversible covalent modification
Enzymes are released into environment in inactive form (zymogen or proenzyme) and peptide bonds are cleaved to become irreversibly activated
- Activation catalyzed by enzymes or environment change
- E.g. pepsinogen is zymogen of pepsin (low pH)
Reversible Covalent Modification
Enzymes activated or deactivated by phosphorylation or addition of some modifier (AMP)
Removal of modifier almost always accomplished by hydrolysis
Phosphorylation occurs in presence of protein kinase
Control proteins
Protein subunits that associate with certain enzymes to activate or inhibit activity
E.g. Calmodulin and G-proteins
Allosteric interactions
Modification of enzyme’s configuration through the binding of an activator or inhibitor at specific binding site on enzyme
Feedback Inhibition
AKA Negative feedback- one of the products downstream in a reaction series inhibits the enzymatic activity of an earlier reaction
Shutdown mechanism when enough product has been produced
Positive Feedback
Occurs less often than negative feedback, but downstream product activates the earlier enzyme in the reaction series
Allosteric inhibitor
A substrate that binds to an allosteric inhibitory site to prevent activity of the enzyme
Substrate does not bind to the active site, but rather a separate site on the enzyme
Binds to enzyme and causes conformational change
- Allosteric regulation
Allosteric regulation
Allosteric inhibition or activation
Binding of a substrate to an allosteric site on an enzyme to cause a conformational change of enzyme and cause either increased or decreased reaction rates
Allosteric Enzymes
Enzymes that have sites for allosteric regulation
At low concentrations of substrate, small increases in concentration increase enzyme efficiency as well as reaction rate
First substrate changes the shape of the enzyme, allowing other substrates to bind more easily (positive cooperativity)
What do enzyme inhibitors effect?
Enzyme inhibitors effect the rate of forward and reverse enzyme reactions
Irreversible Enzyme Inhibitor
Agents that bind irreversibly to enzymes and disrupt function
Usually bind covalently, but can be noncovalent as well
Usually highly toxic
Competitive Inhibitors
Compete with the substrate by binding reversibly with noncovalent bonds to the active site
Bind directly to active site for only a fraction of a second, blocking substrate from binding during that time
Raise apparent K_m, do not change V_max
Reaction rate can be raised to initial V by raising conc. Of substrate to out-compete inhibitor
Uncompetititve Inhibitors
Bind at a site other than the active site
- Only bind to the enzyme-substrate complex
- Substrate remains bound to enzyme once uncompetitive inhibitor binds
- Apparent affinity of enzyme for substrate increases, meaning K_m decreases
- V_max decreases because substrate stays bound longer to enzyme, lowering maximum rate
Mixed Inhibitors
Bind at a site on enzyme other than active site, can bind to either enzyme alone or enzyme-substrate complex
- Most have preference for one state or another
- Can increase K_m if prefer enzyme alone
- Can lower K_m and V_max if prefer E-S
All mixed inhibitors lower V_max to some extent
