BIOCHEMISTRY - CLUTCH REVIEW 2.

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2 CONCEPT: BINDING AFFINITY Protein-ligand binding is reversible, like a chemical equilibrium [S] substrate concentration [E] enzyme concentration Ligands bind to proteins via the same noncovalent forces that dictate protein structure H-bonding, ionic interaction, hydrophobic interaction, and van der Waals forces Allows interactions between ligand and protein to be transient Ligand binding graph θ fraction of protein s ligand binding sites bound to ligand [Ligand] ligand concentration, the units are arbitrary Kd (dissociation constant) concentration of ligand at which ½ the binding sites are occupied - A measure of affinity of a protein for a ligand Page 2

3 CONCEPT: BINDING AFFINITY PRACTICE: Match the dissociation constants to the lines on the graph. Name Kd (M) A 2 x 10-6 B 1 x 10-7 C 1 x 10-6 D 4 x 10-8 E 9 x 10-7 PRACTICE: Which protein binds more tightly to the ligand? What is the dissociation constant of protein 1, and the association constant of protein 2? [Ligand] (nm) θ of Protein 1 θ of Protein Page 3

4 CONCEPT: O2 TRANSPORTATION Hemoglobin (Hb) carries nearly all O2 (99%) Hemoglobin has 4 subunits: each subunit can bind 1 O2 molecule Binding is, subsequent O2 is easier to bind, due to interactions between the subunits Bohr effect hemoglobin's binding affinity oxygen depends on the concentration of CO2 Inverse relationship, so high CO2 and low ph (high acidity) à less oxygen binding Shifts that lower ph favor the T state Shifts that raise ph favor the R state 2,3 Diphosphoglycerate (DPG) a byproduct of glycolysis in erythrocytes, also called 2,3 bisphosphoglycerate (BPG) Increases the amount of oxygen hemoglobin can unload by making hemoglobin go into the T state Shifts O2-Hb curve to the (increases disassociation of oxygen from Hb) Left Shift (Hb has high O2 affinity= low disassociation) Right Shift (Hb has low O2 affinity= high disassociation) PCO2 Acidity (high ph) DPG Temperature PCO2 Acidity (low ph) DPG Temperature Page 4

5 CONCEPT: ENZYMES Enzymes control the rates of reaction Do not affect net G, lower Ea, increasing the reaction rate Do not affect the equilibrium constant, just reach equilibrium faster 5. Isomerase shifts a functional group within the same molecule 2. Transferase (kinase, deaminase) transfers functional groups from one molecule to another 3. Hydrolase (lipase, protease, nuclease) hydrolyzes a bond 1. Oxidoreductase (dehydrogenase, hydroxylase, etc) transfers electrons from reductant to oxidant 4. Lyase (carboxylase, aldolase) makes a double bond 6. Ligase joins two substrates Page 5

6 Michaelis-Menten Model of enzyme kinetics Vo = Vo (initial velocity) y axis [S] (substrate concentration) x axis Vmax maximum velocity of the enzyme (only gets here when substrate concentration reaches saturation) KM (Michaelis constant) concentration of the substrate when half of the binding sites on the enzyme are filled - Substrate concentration at which enzyme is at ½ Vmax (Vo = ½ Vmax) - KM = [S] at ½Vmax Reaction rate = [S]/min, can use rate of disappearance of substrate, or rate of formation of product KM does not depend on enzyme concentration, it is an intrinsic property of the enzyme Vmax varies with enzyme concentration, it is an extrinsic property of the enzyme The Michaelis constant (KM) can be used to compare enzymes and determine their affinity for a substrate. Relative KM is inversely proportional to substrate affinity High KM means low affinity for substrate because it takes a great concentration to reach ½ capacity Low KM means high affinity for substrate because it takes a lesser concentration to reach ½ capacity Enzymes are limited by the rate of diffusion Page 6

7 Lineweaver-Burk equation 1/Vo = (KM/Vmax)(1/[S]) + (1/Vmax) y = mx+b Lineweaver-Burk Plots: y-intercept x-intercept slope Kcat number of substrate molecules converted to product by single enzyme per unit time Called the catalytic constant or turnover number Kcat = Vmax/[enzyme], units are in s -1 Kcat/KM for many enzymes is close to the diffusion-controlled limit Page 7

8 PRACTICE: Use the following data to answer the following questions. [Substrate] (mm) Vo (mm/sec) [Enzyme] (mm) Graph a Michaelis-Menten plot 2. Graph a Lineweaver-Burk plot 3. Determine KM 4. Determine Vmax 5. Determine Kcat Page 8

9 Competitive inhibition the inhibitor and substrate compete for the active site Competitive inhibition increases the amount of substrate needed for a reaction to reach Vmax Affects KM because it competes with the substrate, therefore causes increase in KM Vmax remains the same (NOT changed) Saturation with can overcome competitive inhibition Isosbestic point at x=0 α effect of inhibitor, α = 1 + [I]/Ki [l] concentration of inhibitor Ki binding affinity for inhibitor α = 1 means no inhibitor, as alpha goes up inhibitor concentration increases Uncompetitive inhibition inhibitor binds enzyme-substrate complex Prevents release of substrate Decreases KM and Vmax α (alpha prime) = 1 + [l]/ki (Ki prime) Parallel lines Page 9

10 Mixed inhibition inhibitor can bind to enzyme or enzyme-substrate complex, but doesn t have the same affinity for both Can increase or decrease KM depending on if its affinity for enzyme vs. enzyme-substrate complex Always decreases Vmax TYPE OF INHIBITION X-AXIS INTERCEPT Y-AXIS INTERCEPT None -1/KM 1/Vmax Competitive -1/αKM 1/Vmax Uncompetitive -α /KM α /Vmax Mixed -α /αkm α /Vmax Noncompetitive inhibition: Page 10

11 PRACTICE: Use the following data to answer the following questions. [Substrate] (mm) Vo (mm/sec) Vo w/ inhibitor (mm/sec) What type of inhibitor is this? 2. What is the Ki ([I]=2mM)? Page 11