Kinetic Analysis of Tyrosinase Enzyme

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Kinetic Analysis of Tyrosinase Enzyme

• Experiment 5

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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

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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

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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

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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?

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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

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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
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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.

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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

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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...

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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 ? 475
  nm

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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

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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

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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 100T

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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
  
• (?A/min 0.10-.15)

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• 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
  arent too rapid or too slow)
• If they dont 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

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Kinetic Analysis of Tyrosinase Enzyme

• Experiment 5
• Week2 Determination of Km
• Inhibition

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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

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Competitive Inhibition

• A molecule that fits
• into the enzymes 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

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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

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Uncompetitive Inhibition

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

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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

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Different slopes, same y-intercept (Km for
substrate increases)
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Different slopes, different y-intercept, same
x-intercept (Vmax decreases)
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Same slope, different x-intercept and y-intercept
(Equal change in both Km and Vmax)
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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).

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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 ?A/min 0.033-0.25

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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 100T
• Record absorbance every 30 seconds for 3 minutes
  (blank rate)
• Add the assay volume of tyrosinase, invert,
  immediately set 100T
• Record absorbances every 30 seconds for 4 minutes

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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)

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Data Analysis
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Data Analysis
Prepare a similar table for inhibited
runs ?A/min should be blank corrected
(?A/min)enzyme - (?A/min)blank
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Data Analysis

• Use the following formulas to calculate S and
  vo
• Smg/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 ?c ?A/min (?c
)(.003L)(106 ) vo (umol/min) 3600 x
1cm (?mol/Lmin )
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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)