Enzyme Kinetics SLO's Flashcards
to discuss types of studies you could do with a purified enzyme and what information each type of study provides researchers
Generally studied following purification
■ Protein three-dimensional structure determination
■ Site directed mutagenesis
■ Enzyme kinetics
– Determination of the rate of a reaction and how it changes in
experimental parameters
describe the function and characteristics of enzymes
Biological catalysts ■ Mostly proteins ■ Reaction specific (substrate specific) ■ Structure not changed by the reaction
• Describe the steps in an enzymatic reaction. Be able to calculate the reaction velocity of each step given concentrations and rate constants.
The enzyme and the substrate are in the same area. Some situations have more than one substrate molecule that the enzyme will change.
The enzyme grabs on to the substrate at a special area called the active site. …
A process called catalysis happens. …
The enzyme releases the product.
• Define the initial velocity assumption and discuss its importance in kinetic studies.
The initial linear rate of product formation is called the initial velocity, or vo. It is one of the most important characteristics of any enzyme-catalyzed reaction. One factor that affects the initial velocity is the substrate concentration, [S]
• Define the steady state assumption and discuss its importance in kinetic studies.
Steady state occurs when the rate of formation and breakdown of the intermediate are equal. The steady state assumption relies on the fact that both the formation of the intermediate from reactants and the formation of products from the intermediate have rates much higher than their corresponding reverse reactions.
• Define Vmax
is the reaction rate when the enzyme is fully saturated by substrate, indicating that all the binding sites are being constantly reoccupied.
• Define Km.
is used as a measure of an enzyme's affinity for its substrate. The lower the Km value the higher the enzyme's affinity for the substrate and vice versa Substrate concentration that reaches ½ Vmax ■ Small K m means high affinity ■ High K m means low affinity
• Define kcat
■ kcat - the turnover number
■ E is saturated with substrate
■ kcat has the same units as k2, time-1 (usually s-1)
• Define the kcat/Km ratio. What is the upper limit of this ratio
■ How the enzyme performs when [S] «_space;Km
■ The limit for kcat/Km is the diffusion limit
■ ~109 M-1 s-1
• Define reversible and irreversible inhibitors
An irreversible inhibitor inactivates an enzyme by bonding covalently to a particular group at the active site. A reversible inhibitor inactivates an enzyme through noncovalent, reversible interactions.
• Describe competitive inhibition and how it alters the enzymes kinetics
The competitive inhibitor resembles the substrate and binds to the active site of the enzyme (Figure 8.15). The substrate is thereby prevented from binding to the same active site. A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. Vmax is unaffected structurally similar to substrate when present, 𝐾m of enzyme will increase prevents substrate from binding enzyme reversible by increasing s
• Describe uncompetitive inhibition and how it alters the enzymes kinetics
Inhibitor only binds to ES complex,
when present, 𝐾m of enzyme will decrease
binds enzyme-substrate complex only
end up with parallel lines
• Describe noncompetitive inhibition and how it alters the enzymes kinetics.
In noncompetitive inhibition, a molecule binds to an enzyme somewhere other than the active site Pure equal binding, Vmax changes (reduced) km unaffected unequally binding is mixed
