Functional Characteristics of LDH

Functional Characteristics of LDH Overall Goal To determine the kinetic parameters KM, Vma for LDH and determine Ki for one of the two provided inhibitors of LDH What is the purpose of each of the individual parts? Enzyme Kinetics study of enzyme reaction rates Factors affecting enzyme kinetics: 1. Temp and pH: Each enzymatic reaction has an optimum pH and optimum temperature. Extreme temp or pH disrupts enzyme structure and therefore reaction rate 2. Enzyme concentration: As long as substrate (S) is not limiting, more enzyme (E) leads to more ES 3. Substrate concentration: The rate can be increased by adding more substrate, or by removing product as it is formed 4. Product concentration: Important especially for regulation LDH ENZYME ASSAY COMPARISON Reagent MODULE 3A-D MODULE 4B Enzyme LDH LDH Cofactor NADH NADH Substrate Pyruvate (fixed concentration) Pyruvate (variable concentration) Buffer (pH) Potassium Phosphate (7.4) Tris Diluent Water Water Inhibitor NONE NONE (part A/B) unknown (part C) (8.2) PRELAB WORK: Calculate the amount of total enzyme units you have in your purified LDH sample you are considering to use in this week’s experiment. Discuss with your partner what dilution factor you think you will need to prepare a good assay sample. You will need to have enough enzyme to run at least 30 reactions. Determine if you have enough material for this week’s experiment. If not EMAIL your TFs in advance. MODULE 4B –PART I • Re-assay your LDH using PO4 cocktail. • Calculate activity concentration • Compare this activity concentration to what you had before. • Calculate if you have enough enzyme to set up at least 30 reactions. If you don’t think you have enough consult your TF. LDH catalyzes the last step of anaerobic glycolysis MODULE 4B- PART II • Make a TRIS cocktail using Tris Buffer pH 8.2 • Determine a new “optimal” enzyme dilution using the lowest pyruvate concentration to achieve A340/min=0.02-0.06 • Perform activity assays where you vary [pyruvate] without inhibitor • Initially read absorbance in 5 sec intervals for 2 min. • As [pyruvate] increases the rate of reaction will become rapid. You might need to take more frequent readings and use a shorter monitoring time or reduce the amount of enzyme used. • If you change [enzyme], do NOT forget to correct for this change during data analysis Michaelis-Menten Kinetics Vmax Vmax[S] V= ---------Km+[S] 0.5Vmax KM Lineweaver-Burk plots b y m x MODULE 4B- PART III • Make a NEW TRIS cocktail sing Tris Buffer pH 8.2 and inhibitor. • Make sure to write down the letter and the concentration of your assigned inhibitor. • Perform activity assays where you vary [pyruvate] in the presence of the specific inhibitor. • Make sure to use the SAME total reaction and enzyme volume as you did in uninhibited reactions. • You might need to run the assays longer to see a reliable total absorbance change. Repeat the experiment with the same experiment, changing your roles with your partner. Summary of Different Modes of Inhibition Competitive Uncompetitive Inhibition KmKm Km Non-competitive Un-competitive Summary of the characteristics of each type of inhibition and the reaction scheme Type of inhibition Competitive Uncompetitive Noncompetitive Mixed Reaction Scheme Characteristics The inhibitor binds only to free enzyme. This binding most often occurs in the active site at the precise location where substrate or cofactor also binds. The inhibitor binds only to the enzyme-substrate complex at a location outside the active site. The inhibitor binds equally well to both free enzyme and the enzyme-substrate complex. Hence, these binding events occur outside the active site. These inhibitors display properties of both competitive and noncompetitive inhibition. Ki Kii Ki / Kii finite infinite infinite infinite finite =0 finite finite =1 finite finite >1 Summary of Rate Equations for Different Modes of Inhibition Summary of Double Reciprocal Equations for Different Modes of Inhibition