export_enzymes Flashcards
MM equation assumptions
- Single enzyme
- Single product
- Steady state
- Formation of product is rate limiting
The initial rate of an enzymatic reaction is usually measured by determining the changing concentration of
Product
Characteristic that defines rate of a spontaneous reaction
Difference in energy between the reactant(s) and the transition state
Defines the position of a spont. reaction at equilibrium
Difference in energy between the product(s) and the reactant(s)
An enzyme that is able to act as a general base catalyst of an enzyme-catalyzed reaction
Glutamate
Units for Km
μM
Units for Kcat/KM
μM*s-1
Uncompetitive inhibitors
- Only bind to ES complex
* Uncompetitive inhibitor binds at a site that is different from active site
Describe mixed mode inhibition
- Apparent Km increase, and Vmax decrease
* Equal affinity to bind with E or ES complex
What happens to Kcat/Km in competitive enzyme inhibition
Decrease
What is the best kinetic parameter to compare one enzyme’s utilization of two different substrates?
Kcat/Km
Enzymes increase the rate of reaction by
Dictating the geometry of reactions
When comparing a catalyzed and an uncatalyzed reaction, what changes
- ΔG
* Initial rate of rxn
How do competitive, uncompetitive, and mixed mode inhibitors differ in their mode of action?
- Competitive : binds to enzyme at active site
- uncompetitive : binds to enzyme-substrate complex at other site
- mixed : bonds to enzyme or enzyme-substrate complex at other site
Chymotrypsin belongs to a group of proteolytic enzymes called the “serine proteases,” many of which have an Asp, His, and Ser residue that are crucial to the catalytic mechanism. The serine hydroxyl functions as a nucleophile. What do the other two amino acids do to support this nucleophilic reaction?
- histidine functions as a general base, accepting a proton from the serine hydroxyl, thereby increasing serine’s reactivity as a nucleophile.
- The negatively charged Asp stabilizes the positive charge that develops on the His.
Chymotrypsin cleaves on the C-side of amino acid residues with flat, nonpolar side chains. Trypsin cleaves on the C-side of arginine and lysine, while elastase cleaves on the C-side of glycine and alanine. What amino acids in the structure of the enzymes account for these differences?
Chymotrypsin has a deep hydrophobic pocket. The binding pocket in trypsin has an aspartate at the bottom, while elastase has bulky valine and threonine causing a shallow pocket.
What kind of covalent intermediate is involved in the aldolase mechanism? What amino acid side chain is involved?
Imine (Schiff base) formed with lysine
What metal ions are involved in the enolase mechanism and what role do they play?
Mg2+. Stabilize the enolate thus making the C-H more acidic.
What is the standard way to compare enzyme efficiencies?
specificity constant – kcat/Km
Carboxypeptidase A will not remove the C-terminal amino acid if it is Lysine or Arginine, while carboxypeptidase B will only remove terminal Lys or Arg. What amino acid would you expect to be present in the binding pocket of carboxypeptidase B?
Aspartate or glutamate
Competitive, uncompetitive, and mixed mode inhibitors are all types of reversible inhibitor. What do irreversible inhibitors do?
Bind covalently with or destroy a functional group that is essential for the enzyme’s activity.
Kcat/Km is a measure of
an enzymes catalytic efficiency, also called the specificity constant – best way to compare two different enzymes or two different substrates for the same enzyme
Kcat is a general rate constant, also called the
turnover number , that describes the limiting rate in any enzyme catalyzed reaction at saturation
