6. Enzyme Kinetics Flashcards
Why is Enzyme Kinetics Information Useful?
To estimate the biological activity of an enzyme
To estimate of the cellular concentrations of metabolites
To compare healthy and disease states
For the characterization of inhibitors
Michaelis-Menten Model
v=k3[ES]
Km= (k2+k3)/k1
E+S -> ES -> E+P
E=enzyme, S=substrate, P=product, ES=enzyme-substrate complex, k=rxn rate constant
K1, k2, k3?
K1 (E+S -> ES)
K2 (ES -> E+S)
K3 (ES -> E+P)
Michelis-Menten model ignores the reverse reaction
Velocity (v) of forward reaction is directly proportional to [ES]
KM is the ratio of rate constants of the breakdown and formation of ES, a.k.a. Michaelis Constant
Michaelis-Menten Equation
(pic)
v = (Vmax[S]) / (Km + [S])
M-M graph
X-axis: [S]
Y-axis: velocity (v)
Vmax and KM are difficult to accurately ascertain from a Michaelis-Menten graph
Lineweaver-Burke
Transformation
(pic- graph)
1/v= (Km/Vmax)(1/[S]) + 1/Vmax
Slope= Km/Vmax x-int= -1/Km y-int= -1/Vmax
Vmax = maximal velocity KM = substrate concentration at half maximal velocity
Michaelis constant (KM)
Substrate concentration when reaction velocity is ½ Vmax
Characteristic of an enzyme and its particular substrate
Constant for an enzyme isoform
A measure of the affinity of an enzyme for its substrate
-High KM – Low affinity
-Low KM – High affinity
Applies to the natural substrate of an enzyme
Does not vary with the concentration of the enzyme
One reaction, two enzymes
(pic- graph 1. Hexokinase In most tissues Low KM (high affinity) Low Vmax For normal metabolism of glucose
2. Glucokinase In liver & pancreas High KM (low affinity) High Vmax For clearing glucose from blood when levels are high (After a meal)
Comparison of the activities of hexokinase and glucokinase. The Km for hexokinase is significantly lower (0.05mM) than that of glucokinase (10mM). This difference ensures that non-hepatic tissues (which contain hexokinase) rapidly and efficiently trap blood glucose within their cells by converting it to glucose-6-phosphate. One major function of the liver is to deliver glucose to the blood and this in ensured by having a glucose phosphorylating enzyme (glucokinase) whose Km for glucose is sufficiently higher that the normal circulating concentration of glucose (5mM).
Kinetic parameters
Vmax- depends on amount of enzyme [Et]
Kcat = (the turnover number)
Kcat = moles product formed per s per mole of catalytic center
Unit of enzyme activity, e.g., mmoles product/min
Specific Activity = Units/mg protein
Factors that affect
enzyme activity
Substrate concentration Enzyme concentration Temperature pH Activators/inhibitors
Effect of [Substrate]
The rate of an enzyme catalyzed reaction increases with substrate concentration
This increase in rate occurs until maximum velocity (Vmax) is reached
Vmax reflects the saturation of all the available binding sites on the enzyme with substrate
Enzymes following M-M kinetics show hyperbolic curve
Allosteric enzymes show sigmoid curve
Effect of enzyme concentration
Reaction velocity is directly proportional to the enzyme concentration when the substrate concentration is unlimited
At a given substrate concentration, the initial velocity of an enzyme catalyzed reaction is proportional to the enzyme concentration
A property made use of in determining the amount of an enzyme in plasma, serum or tissues
v vs [E] graph is directly proportional
Effect of temperature
Reaction velocity increases with an increase in temperature till a peak is reached and then decreases with further increases in temperature
Optimum temperature for most human enzymes is 35-40◦C
*Thermostable DNA polymerases are an exception to this rule
Effect of pH
Changes in pH affect ionic charge of amino acid side chains of enzymes (charged amino acids) and dramatically affect catalytic function
Most enzymes in the human body function optimally in the physiological pH range (around pH 7.4)
Some Enzymes have different optimum pH
-Pepsin: 1-2
-Acid Phosphatase: 4-5
-Trypsin: 6-7
-Alkaline phosphatase: 9-10
Enzyme inhibition
Reversible inhibition: Non-covalent attachment of the inhibitor
- Competitive (common)
- Noncompetitive (common)
- Uncompetitive (rare)
- Allosteric modulation (mainly multisubunit enzymes)
Irreversible inhibition: Covalent attachment of the inhibitor (also called suicide inhibition)
Reversible inhibition- 1. Competitive Inhibition
A competitive inhibitor mimics the substrate
Increasing the amount of substrate overcomes the inhibition
Kinetics
(graph)
Michaelis constant, Km, inc in presence of competitive inhibitor
Maximal velocity, Vmax, same in presence of competitive inhibitor
Clinical Drugs
1. Sulfanilamide and its derivs competitively inhibit syn of folic acid in microorganisms and thus dec syn of nucleotides needed for replication
- Dihydrofolate reductase is completely inhibited by methotrexate (treat cancer of the blood, bone, lung, breast, head, and neck), a folic acid analogue used to effect remission of acute leukemia in children
-Competitive Inhibitors Mimic Substrates
»methotrexate mimics folate - Statins (e.g., Lovastatin, Simvastatin) - Reduce plasma cholesterol levels by inhibiting hepatic cholesterol synthesis (attach at HMG CoA reductase active site)
HMG-CoA reductase is the rate-limiting enzyme of cholesterol biosynthesis
Reversible inhibition-
2. Noncompetitive inhibition
Inhibitor binds at a site other than the substrate binding site (active site)
-eg binds at inhibitor site and alters structure of active site; substrate molec may still bind w enzyme molec but rxn is hindered
Prevents catalytic activity
A noncompetitive inhibitor can bind either to the free enzyme (E) or to the enzyme-substrate complex (ES)
The ESI complex cannot produce the product (inactive)
Increasing [S] cannot overcome the inhibition
Kinetics
(graph)
Michaelis constant, Km, unchanged in presence of noncompetitive inhibitor
Maximal velocity, Vmax, is dec in presence of noncompetitive inhibitor
eg
Acetylcholine esterase (neurotransmission)
-Acetylcholine (substrate)
-noncompetitive inhibitor=physostigmine
Ferrochelatase (Heme biosynthesis)
Fe2+ (substrate)
-Pb (noncompetitive inhibitor)
