Enzyme Kinetics 12 Flashcards
Rates of enzyme catalyzed reactions
- enzyme kinetics: used to determine quantitative relationships
- a progressive curve is used to measure a product formation (or substrate loss) as a function of time
- velocity is linear at first, but may decrease due to product inhibition as equilibrium is approached, or by enzyme inactivation
- the rate of product formation will generally increase with the amount of enzyme present (assay)
Rate equations
-a unimolecular reaction has a velocity rate that is dependent on the concentration of only one substrate
V=k[A], where the rate constant k has unit sec^-1
-a bimolecular (second order) reaction has a velocity that is dependent on 2 substrat concentrations
V=k[A][B], where k is M^-1 sec^-1
Velocity vs substrate conc
Velocity vs [S] curve is hyperbolic
-dependent upon rate determining step
Michaelis-Menten Equation
V=Vmax[S]/Km+[S]
Rate vs efficiency
-catalytic rate constant determines how quickly and enzyme can act, while Kcat/Km determines catalytic efficiency
-Kcat = catalytic rate constant/turnover number. The number of catalytic cycles that each active site undergoes per unit time, when the enzyme is saturated with [S]
Kcat=Vmax/[Etotal]
-Kcat often equivalent to K2 (RDS)
-catalytic efficient reflects enzyme rate and substrate affinity
-enzymes reach catalytic perfection when their rate is diffusion controlled (react as quickly as they are available)
Kcat/Km=10^8 or 10^9
Lineweaver-Burk ploy
- linearizes the Michaelis-Menten kinetics data
- takes reciprocal of both sides
Reversibility of enzyme inhibition
- enzyme inhibitors are important in medicine and biotechnology, and as research tools
- irreversible inhibitors usually modify the active site covalently and cannot be reversed
- Transition state analogs often make better inhibitors than substrate analogs (tighter binding to active site)
- Reversible inhibitors normally bind to enzymes non-covalently, and can be classified by where they bind and what reaction steps they block; these are kinetically distinguishable:
- competitive
- non-competitive
- uncompetitive
- mixed etc…
Competitive Inhibitors
- increase Km
- substrate and inhibitor bind at same site and are mutually exclusive.
- large excess of S can overwhelm I
- Km increased
- Vmax unchanged
Non-competitive inhibitors
- decrease Vmax
- S and I are not mutually exclusive, but ESI complex is less active than ES
- Km unchanged
- Vmax decreased
Other types of inhibition
-mixed: similar to non-competitive but binding to stand site modifies Vmax and Km
- uncompetitive: inhibitors bind to enzyme after substrate binds
- both Vmax and Km are reduced by same amount
Exceptions to Michaelis-Menten kinetics
- not all enzymes follow Michael is-men ten kinetics
- many require multiple substrates/products and/or require multiple steps (Chymotrypsin)
-multistep reactions: Km is a complicated function of many rate constants
Allosteric enzymes
- often have multiple subunits and show cooperativity (interaction among subunits produces signmoidal rather than hyperbolic substrate curves (analogous to Hb vs Mb)
- allosteric affections may inhibit or activate
- this often involves and early and/or committed step in a metabolic pathway
Clinical connection: drug design to protein kinases
- protein kinases: a family of >500 proteins involved in signalling pathways (and are drug targets for many diseases)
- the problem: the active site is very similar for many kinases, making selective drug design difficult
- the solution: design inhibitors to the allosteric site, which is more unique to each enzyme
