1. Kinetic Analysis of Tyrosinase Enzyme Experiment #5

2. Enzymes as catalysts • It is necessary for biological reactions to occur much quicker than the ambient temperature and prevailing conditions would allow • “catalyst” a substance that when added to a chemical reaction, speeds it up without altering the final products or without itself being consumed. • Enzymes are biological catalysts

3. Enzyme Benefits • Enzymes provide many medical benefits • key to understanding inborn errors of metabolism • important in detoxification reactions • targets of chemotherapy • aid in diagnosis and monitoring therapy • primary role of vitamins is as enzyme cofactors • key to metabolic control and balance

4. Enzyme properties • All enzymes are proteins • Molecular Weight range: 15 kd-1000 kd • enzymes show the same physical and chemical properties as all proteins • denaturation • precipitation • sensitivity to proteases • Enzymes are efficient biological catalysts which must operate at 37o C or below and at pH values found in living cells

5. Enzyme Properties • Enzymes are highly specific in their catalysis • they must bind (form a complex) with substrate into a region of the enzyme known as the “active site” • How?

6. Enzyme Properties • Enzymes also allow the regulation of reactions through activation or inhibition of the enzyme by effectors • ** Virtually all biological reactions are found to be enzyme catalyzed**

7. Enzyme Kinetics • Studies of enzyme kinetics began in 1902 by Adrian Brown • studied the rate of hydrolysis of sucrose • proposed that the overall reaction was composed of two elementary reactions The enzyme-substrate complex (ES) provides the transitional state that facilitates a more rapid production of products

8. Enzyme Kinetics • In 1913, Lenor Michaelis and Maude Menten made the assumption that the reversible step in the mechanism does achieve equilibrium • Therefore, rewriting the law of chemical equilibrium for the reversible step and equating the ratio of the forward to reverse rate constants and making substitutions….

9. Enzyme Kinetics Michealis-Menten equation vo = Vmax [S] Km + [S] Vmax= the rate of reaction in which all of the active sites of the enzyme are consumed by substrate Km= a ratio of all rate constants involved. Km also represents the substrate concentration at which the reaction rate is 1/2 of Vmax [S] = the concentration of substrate binding to enzyme

15. Effects of Temperature on Enzymes • Living systems must function in a relatively restricted range of temperature • Enzyme catalyzed reaction rates will increase with temperature • will approximately double for every 10o increase • However, enzymes are proteins…...

17. Tyrosinase Enzyme • Copper containing oxidase • Widely distributed in plants, animals, and humans • In plant cells, is responsible for browning in potatoes, apples and bananas • In human cells, is responsible for catalyzing the biosynthesis of melanin pigments, causing suntans • Method of assay: Tyrosinase + (d,l)-Dopa  Dopachrome λ= 475 nm

18. Kinetic Assay Procedure • Overview: • DETERMINE TOTAL CONCENTRATION OF ENZYME • DETERMINE IDEAL LEVEL OF TYROSINASE FOR KINETIC ASSAYS • PERFORM KINETIC ASSAYS TO DETERMINE Km • INHIBITION OF ENZYME ACTIVITY

19. Kinetic Assay Procedure • Week #1: • Estimate enzyme concentration • Determine ideal volume of enzyme to use • Determine appropriate substrate volume range • Determine Tyrosinase Concentration • adjust UV-VIS to 280 nm • zero out instrument using 0.05M pH 7.0 phosphate buffer • measure absorbance of tyrosinase solution • Calculate concentration assuming a 1% w/v standard has an absorbance of 24.9

20. Kinetic Assay Procedure • ** All reagents except enzyme will be stored at room temperature** • Determination of Ideal Enzyme Volume: • Initially set up all 5 assays as given in the table EXCEPT for adding enzyme (gently invert to mix) Reagent (mL) 1 2 3 4 5 phosphate buffer 1.45 1.40 1.30 1.20 1.10 l,d-Dopa 1.5 1.5 1.5 1.5 1.5 Tyrosinase 0.05 0.10 0.20 0.30 0.40 • Place into spectrophotometer at 475 nm and immediately set 0 and 100%T

21. Kinetic Assay (continued) • Record absorbance every 30 sec for 3 minutes (“Blank Rate”) • Add the assay volume of tyrosinase (invert to mix) • Record absorbances every 30 seconds for 4-5 minutes • Choose the enzyme volume that provided a convenient rate at saturating levels of substrate (ΔA/min = 0.10-.15)

22. Determination of Substrate Volume: Designed as a trial run to make sure the recommended volumes of substrate concentration will work (sufficient changes in absorbance that aren’t too rapid or too slow) If they don’t work, formulate a volume range that will **Set up assays containing fixed volume of enzyme. Total volume always 3.0 mL** Reagent 1 2 3 4 5 phosphate buffer (3.00 - (substrate + enzyme) d,l-Dopa 0.10 0.40 0.80 1.0 1.50 tyrosinase *optimal enzyme volume *Run assays just like previous step..running a blank, adding enzyme last, recording measurements every 30 sec

23. Kinetic Analysis of Tyrosinase Enzyme Experiment #5 Week#2: Determination of Km Inhibition

24. Enzyme Inhibition • Inhibitors can halt the activity of an enzyme • results in a decreasing concentration of product formation • Drug therapy is based on the inhibition of specific enzymes • There are three major classes of inhibitors • Competitive • Noncompetitive • Uncompetitive

25. Competitive Inhibition • A molecule that fits into the enzyme’s active site but does not react with it • Enzyme will remain inactive until the inhibitor falls off • More substrate is needed to get to the maximum rate, since substrate “competes” with inhibitor

26. Noncompetitive Inhibition • Inhibitor fits into a site on the enzyme different from the active site • As a result, the folding of the enzyme changes a bit, distorting the active site in a way that makes it less effective as a catalyst • A decrease in the maximum rate would be observed since each catalyst has become less efficient

27. Uncompetitive Inhibition • Inhibitor binds to the enzyme only after enzyme-substrate complex forms • As a result, catalytic activity is blocked

28. Irreversible Inhibition • Inhibitor may bind to the active site or alternative site • Next, inhibitor forms a covalent bond to the enzyme • Since inhibitor, will not fall off, the enzyme molecule is dead

29. Different slopes, same y-intercept (Km for substrate increases)

30. Different slopes, different y-intercept, same x-intercept (Vmax decreases)

31. Same slope, different x-intercept and y-intercept (Equal change in both Km and Vmax)

32. Inhibitors to tyrosinase • Several compounds act as inhibitors to tyrosinase enzyme….. We will examine: • Thiourea • Cinnamic Acid “Cinnamic acid was found to be effective in apple juice, especially when used in combination with ascorbic acid (Walker, 1976: Sapers et al., 1989b). This inhibitor was also effective when applied to cut surfaces of apples, but induced browning under some circumstances. Carbon monoxide has been proposed as a browning inhibitor for mushrooms (Albisu et al., 1989).”

33. Procedure • I. Determination of Km • Set up the following assays using ideal volume of enzyme and ideal substrate range. Total volume is 3.0 mL Reagent 1 2 3 4 5 phosphate buffer (3.00 - (substrate + enzyme) d,l-Dopa 0.10 0.40 0.80 1.0 1.50 ** tyrosinase *optimal enzyme volume ** substitute substrate volume range that worked best** Ideal product formation is ΔA/min = 0.033-0.25

34. Procedure • Set up all 5 assays as given in the table except for adding the enzyme • Place into spectrophotometer at 475 nm and immediately set 0 and 100%T • Record absorbance every 30 seconds for 3 minutes (“blank rate”) • Add the assay volume of tyrosinase, invert, immediately set 100%T • Record absorbances every 30 seconds for 4 minutes

35. Procedure • II. Inhibition • Choose one of the three inhibitors • Choose a constant level of inhibitor that results in at least 20-30% decrease in rate (for an intermediate concentration of substrate) Reagent 1 2 3 4 5 phosphate buffer (3.00mL - (substrate + inhibitor + enzyme)) d,l-Dopa 0.10 0.40 0.80 1.0 1.50 inhibitor ** determined by trial and error tyrosinase *optimal enzyme volume Run assays just like in Km determination (set up blank rate with buffer, substrate, and inhibitor)

36. Data Analysis

37. Data Analysis Prepare a similar table for inhibited runs **A/min should be blank corrected: (ΔA/min)enzyme - (A/min)blank

38. Data Analysis Use the following formulas to calculate [S] and vo [S]mg/mL = (volume used in assay)(“Stock conc.” mg/mL) 3.0 mL [S] mol/L = Smg/mL x 1 197.2 The rate of reaction is dependent on production of Dopachrome molar absorptivity constant of dopachrome = 3600 mol/Lcm Δc = ΔA/min (Δc )(.003L)(106 ) = vo (umol/min) 3600 x 1cm (Δmol/Lmin )

39. Data Analysis • Use linear regression to calculate slope and intercept for inhibited and uninhibited plot • Uninhibited Inhibited y-intercept= 1/Vmax compare slope and 1/y-intercept = Vmax y-int to inhibited competitive & noncompetitive slope = Km/Vmax slopeinh = slopeuninh (1 + [I]/KI) Km = (slope)(Vmax) [I] = (ml inhibitor used)(inhibitor, M) (3.0 mL) uncompetitive y-interceptinh = y-interceptuninh (1 + [I]/KI)