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 How?
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 Km + [S]
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)
phosphate buffer
l,d-Dopa
Tyrosinase
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 = )

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
phosphate buffer ( (substrate + enzyme)
d,l-Dopa
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
phosphate buffer ( (substrate + enzyme)
d,l-Dopa **
tyrosinase *optimal enzyme volume
** substitute substrate volume range that worked best**
Ideal product formation is ΔA/min =

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
phosphate buffer (3.00mL - (substrate + inhibitor + enzyme))
d,l-Dopa
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 [S]mg/mL = (volume used in assay)(“Stock conc.” mg/mL)
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)