Biochemistry I Lecture 6 Spring/Summer 2024 PDF

Biochemistry I Spring/Summer 2024 Lecture 6 April 16, 2024 Textbooks: Mark’s Basic Medical Biochemistry 5th Edition And Harper’s Illustrated Biochemistry 30th Edition Read Chapters 8-11 in Mark’s Basic Medical Biochemistry Last Time: Enzymes are catalysis that vastl...

Biochemistry I Spring/Summer 2024 Lecture 6 April 16, 2024 Textbooks: Mark’s Basic Medical Biochemistry 5th Edition And Harper’s Illustrated Biochemistry 30th Edition Read Chapters 8-11 in Mark’s Basic Medical Biochemistry Last Time: Enzymes are catalysis that vastly accelerate the rate of a biochemical reaction. Enzymes lower the activation energy by: Bringing 2 or more reactants into close proximity and placing them in a favorable orientation. Stabilization of the charged transition state. General acid-base catalysis/covalent catalysis/metal ions which act as Lewis acids (electron pair acceptors)  Hexokinase (other tissues) GLUT Glucokinase (liver) Glucose → Glucose → Glucose 6-phosphate Liver RBC, CNS glycogen glycolysis glucose pyruvate Glucose (released to the bloodstream) glucose ATP Glut II Liver, β cells of the pancreas, kidney High capacity, low affinity Glucose sensor in β cells of the pancreas “Two –Way” transporter Liver hepatocytes need two-way transporters for uptake and release of glucose. β cells of the pancreas need to monitor blood glucose levels precisely GLUT I RBC and III CNS (high affinity) Glut III major CNS glucose transporter glycogen glycogenolysis glucose glucose gluconeogenesis  1. The initial rate for an enzyme catatyzed reaction which apparently obeys Michaelis-Menton kinetics is determined at a number of substrate concentrations: [S] μmol/L v (μmol/L) min-1 5 22 10 39 a. estimate Vmax and Km from the plot 20 65 50 102 Vmax ≈ 100 120 200 135 Km ≈ v vs [S] 160 140 Vmax S 120 v 100 K M  S 80 60 40 20 kcat [E]t = Vmax 0 0 50 100 150 200 250  b. From the data above, 1/v vs 1/[s] is plotted below. Calculate Vmax and Km from the Vmax = equation y = 0.1961x + 0.0061 1/[S] 1/v Km = 0.2 0.045455 0.1 0.025641 x intercept = ( -1/Km) = 0.05 0.015385 0.02 0.009804 pay attention to units: [S] is in μmol/L, v 0.01 0.008333 is in μmol/(L min), 0.005 0.007407 c. verify that Km/Vmax has units of 1/v vs 1/[S] time, in this case, minutes. 0.06 y = 0.1961x + 0.0061 R² = 0.9995 0.04 0.02 0 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 -0.02 -0.04 -0.06 d. If the total enzyme concentration used in this experiment was 1.0 nmol/L, how many molecules of substrate are produced per minute? In other words, calculate the turnover number kcat where Vmax = kcat [E]T (1000 nmol = 1 μmol) e. How many molecules are produced per second? ( 60 s = 1 min ) f. Calculate kcat/Km in L/(mol s) (kcat/Km) has units of L /(μmol min). Which enzyme from lecture 5 notes is similar in efficiency? g. from the equation y = 0.1961x + 0.0061 and your values for Vmax and Km – construct the Michaelis-Menton equation. 2. In an enzyme reaction, Vmax was found to be 75.5 μM/min when 23 μL of a 29 nM enzyme stock solution was used in a total volume of 3 mL. Calculate kcat in units of s-1 60 seconds = 1 minute  1 0.007 0.06 2 0.015 0.11 3 0.031 0.16 4 0.068 0.21 5 0.1 0.23 6 0.2 0.28 7 0.3 0.29 8 0.4 0.28 V (ADH) vs [ethanol] 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Liver alcohol dehydrogenase (ADH) catalyzes the oxidation of ethanol (shown in red) to acetaldehyde. The active site of liver ADH contains a bound zinc atom, and a serine side chain –OH, and a histidine nitrogen that participate in the reaction. The histidine pulls an H+ off the active-site serine, which pulls the H+ off of the substrate –OH group, leaving the oxygen with a negative charge that is stabilized by zinc.  Alcohol dehydrogenase Acetaldehyde (ADH) dehydrogenase Ethanol → acetaldehyde → acetate NAD+ → NADH Formepizole: methanol poisoning, ethylene glycol poisoning. A Disulfiram: competitive competitive inhibitor of inhibition ADH NADH/NAD+ Lactic level increases acidosis Fasting hypoglycemia Fatty liver Hepatic steatosis The mechanism of aldehyde dehydrogenase via a tetrahedral thiohemiacetal intermediate. The active residues, Cys302 and Glu268, catalyze the reaction  Competitive inhibition https://bio.libretexts.org/Bookshelves/Biochemistry/Supplemental_Modules_%28 Biochemistry%29/6._Lab_Notes_Part_2/6.2%3A_Enzyme_kinetics