Chapter 7 - Kinetics and Regulation
Terms in this set (29)
What is Kinetics?
Study of reaction rates
What types of reactions are there?
First ordered reaction (A->P) rate expressed as function of time - dependent on substrate
Second ordered reaction (SA->P OR A+B->P)- dependent on two substrates
What are Pseudo and zero order reactions?
Pseudo - second order reactions appearing as 1st order
eg. If [B] greatly exceeds [A] or [A] is low the reaction rate will be first order in respect to A and not dependent on B
Zero order reactions - independent of reactant concentration
How do you display Kinetic Rates?
On a graph of Product vs Time - enzyme acitivty rates typically graphed as intial velocity vs amount of substrate
Intial velocity determined from intial slope of reaction rate vs time
Intial velocity then determined from diff substrate concentrations then plotted
What is the Michaelis-Menten Model?
Km=(Kneg1 + K2) / K1
Vintial = Vmax x [S] / [S]xKm
-Enzyme + Substrate - ES - Enzyme + Product
-E+S-ES is reversible
-At intial velocity - Product concentration is very low and diffuses away (not converted back to substrate)
-K1-rate constant for formation of ES
-K2-constant for formation P
-Kneg1&2-constants for respective reverse reactions
Why is Vmax important for reaction velocity?
Vmax is only attained when all the enzyme is bound to S - it is directly dependent on enzyme concentration
-it is the velocity of reaction with plateau at certain substrate concentration and after this point and increase in S concentration results in no further increases in Velocity
What are the characteristics of Km - Michaelis constant?
-Km represents the substrate concentration required for half max activity - characteristic of the enzyme
-it is also considered the afffinity constant but inverse - a higher Km means less affinity
-dependent of particular substrate and environmental conditions eg pH, temp, and ionic strength
-Km=[S] at which half active sites are filled - provides measure of [S] required for significant catalysis to take place
What does Km tell us about enzyme sensitivity?
At values below Km - enzymes sensitive to changes in [S] but display little activity
At values above Km - enzymes have great catalytic activity but insensitive to changes in [S]
What are isoenzymes?
enzymes that differ in amino acid sequence but catalyze same reaction, but - different tissues, kinetics and regulation
eg. Glocokinase - traps glucose in the liver when there is exess in the blood
-high Km therefore low affinity so it does not want first access to glucose
Hexokinase - low Km, lower Vmax - inhibited by glucokinase
What is a Single Nucleotide Polymorphisms?
small mutation that changes kinetics of enzyme
eg. Acetaldhyde Dehydrogenase - known mutation with higher Km - results in acetylhyde accumulation - causes 'alcohol flush'
What is a sequential reaction?
-Subsrate NADH binds to enzyme followed by second substrate Pyruvate to form ternary complex of 2 product plus enzyme
-products subsequently leave at same time
What is a Double Displacement reaction?
First substrate binds and first catalytic step takes place resulting in subsituted enzyme (e-NH3)
-first product then leaves
Second substrate binds to subsituted enzyme and second catalytic step takes place - the NH3 is transferred to substrate to form final product which departs the enzyme
What is the difference between Sequential and Double Displacement reactions?
S.R - all substrates must bind to enzyme before any product is released
D.D - one or more products is released before all substrates bind to enzyme
-there is also existence of a substituted enzyme intermediate - enzyme temporarily modified
What are the characteristics of Allosteric enzymes?
Allosteric enzymes are regulated enzymes that are required to coordinate metabolism to control metabolite flux
-they regulate flux of biochemicals through metabolic pathways and regulate catalytic activity by environmental signals including final product of metabolic pathway
What is a metabolic pathway?
A series of enzymatic reactions - control of key enzymes (committed steps) controls pathway
-1st step always committed step - control of initiating the pathway
-When sufficient product is present - it can bind reversibly to the first enzyme cataclyzing the committed step and inhibit the reaction (Feedback inhibition) - will bind to regulatory site on e1 - regulatory site distinct from active site
What is Allosteric Regulation?
Provides immediate response to metabolic challenges
-Allosteric enzymes can recognize inhibitor molecules as well as stimulatory molecules
Kinetics of Allosteric Enzymes
Allosteric enzymes display sigmoidal dependence of reaction velocity on substrate concentration in contrast to hyperbolic curve seen with Michaelis Menten enzymes
-Sigmiodal indicated intial velocity is relatively slow at low substrate concentration then speeds up before reaching Vmax plateau
Allosteric vs M-M enzymes
Allosteric Enzymes distinguished by their response to changes in substrate concentration and susceptibility to regulatio by other molecules
-they are more sensitive to substrate conc. especially near Km which allows for more sensitive control of reaction velocity
-sigmodial curve also represents multiple active sites on the allosteric enzymes as well as regulatory sites
What are the two models of function for the activation E and sigmiodal kinetics of Allosteric Enzymes
Concerted and Sequential models
What are the features of the Concerted Model?
-The enzyme exists into 2 quaternary structures designated T(tense) and R(relaxed)
-The R state is enzymatically more active then the T state
-The is the more stable T state but binding of a substrate causes a shift to R state
-All active site must be in the same state
What are the features of the Sequential Model?
-R state enzymatically more active then T-state
-T and R states can exist as hybrids
-binding at one site influence binding at another site - eg increases affinity for next binding
Concerted vs Sequential models
-A.E have multiple active sites on different polypeptide chains
-enzyme can exist in 2 distinct conformations/states - in absence of substrate/signal molecules R and T are in equilibrium
-Binding disrupts this equilibrium in favour of R
-threshold effect - once threshold reached activity rapidly increases
-subunits of A.E undergo sequential changes in structure
-binding influences binding to neighbour without inducing transition encompassing entire enzyme
-accommodates neg cooperativity - binding of 1 substrate decreases affinity of other sites for the substrate
What is the T to R transition state
The shift from T to R results in a narrow range with steep slow - allosteric more "switchlike"
-regulating molecules alter the equilibrium between T and R forms - change relative proportion of enzymes in T n R states
What is the difference between pos n neg effector and how does this influence the T to R transition state?
-Pos effector binds to R form at regulating site, difference from active site, and stabilizes this form increasing R concentration and making R and substrate interaction more likely
-Neg effector binds to T and stabilizes in increasing concentration of T therefore decreasing likelihood of R binding to substrate
What effect does each effector have on the Substrate concentration threshold?
Pos effectors decrease threshold of substrate concentration needed for activity whereas neg effectors increase threshold
What are heterotropic effects? Homotropic?
-The effects of regulatory on allosteric enzymes
-shifts sigmiodal curve left (activators) or right (inhibitors)
-the effects of substrates on allosteric enzymes
-account for sigmiodal nature of kinetics cuve
Homotropic vs Hetertropic regulators
-substrate acts as allosteric effector for another binding site
-effector distinct from substrate - metabolites of process (up or down stream of reaction)
What is Gout?
It is a clinical example of faulty allosteric regulation
-failure of feedback inhibition of PPS, enzyme involved in synthesis of purines
-excess purines broken down resulting in increase urate - excess urate accumulates and crystallizes in joints leading to inflammation
What is Enzyme Heterogenity?
ability of molecule, with passage of time, to assume several different structures that differ slightly in stability
-single molecule studies can reveal molecular heterogenity