Kinetic Analysis Of Tyrosine Biology Essay

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Enzymes catalyze reactions in physiological systems.  In an equilibrium, an enzyme (E) binds a substrate (S) to form an enzyme-substrate complex (E-S).  The E-S complex can dissociate or irreversibly convert the substrate to a product (P) (Scheme 1).  The Michaelis-Menten equation describes the relationship between the rate of substrate conversion by an enzyme to the concentration of the substrate (Equation 1).  In this equation, V is the rate of conversion, Vmax is the maximum rate of conversion, [S] is the substrate concentration, and Km is the Michaelis constant, the substrate concentration at which the rate of conversion is half of Vmax.  A more illustrative version of the Michaelis-Menten equation is the Lineweaver-Burk equation (Equation 2).  The Lineweaver-Burk equation affords a line with a slope of Km/Vmax and y-intercept of 1/Vmax.  The x-intercept, a theoretical point since 1/[S] cannot be negative, is -1/Km.

Scheme 1 - enzyme-substrate complex and product formation model

Enzyme inhibition is a common goal for the pharmaceutical industry.  All inhibitors cause the substrate to react at a lower rate than without the inhibitor.  Reversible enzyme inhibitors fall into three categories - competitive, non-competitive, and uncompetitive.  Furthermore, non-competitive inhibitors can be divided into two additional categories - pure and mixed.  The Lineweaver-Burk equation can be used to categorize different inhibitors.  Understanding the type of inhibitor will give clues on how its structure might be modified to increase its potency.

Competitive inhibitors bind at the active site of the enzymes to form an E-I complex (Scheme 2).  The inhibitor blocks the active site, and the substrate cannot bind until the inhibitor dissociates.  Since the inhibitor and substrate compete for the same site, raising the substrate concentration can eventually overcome the inhibitor, and Vmax can be achieved.  Although Vmax can be reached, a competitive inhibitor raises Km, indicating that the affinity of the enzyme for the substrate is lower in the presence of the inhibitor.  The effect of a competitive inhibitor in a Lineweaver-Burk plot is both to move the x-intercept and increase the slope.  Plots made with varying amounts of a competitive inhibitor will all cross at the same y-intercept.

Scheme 2 - competitive inhibitor model

Non-competitive inhibitors bind at an allosteric site on the enzyme and leave the active site unblocked.  In a pure non-competitive system, the substrate has an identical affinity for both the E-I complex and enzyme.  Unlike the E-S complex, the E-I-S complex cannot convert the substrate to product (Scheme 3).  With a pure non-competitive inhibitor, the Km value is unchanged while Vmaxis lowered.  So, the x-intercept will be constant, and the slope will increase with more inhibitor.  Note that pure non-competitive inhibitors are virtually unknown.  With a mixed non-competitive inhibitor, the affinity of the E-I complex for the substrate is not the same as the unbound enzyme.  In this case, not only is Vmax lowered, but Km is also raised.  The Lineweaver-Burk plot will show changes in the x-intercept and increasing slope.

Scheme 3 - non-competitive inhibitor model

Uncompetitive inhibitors are thought to bind the E-S complex and not the enzyme.  As with non-competitive inhibitors, the E-S-I complex cannot form the product.  The product can only be formed from the E-S complex (Scheme 4).  The effect of an uncompetitive inhibitor is to decrease both Vmax and Km.  The drop in Km deserves some comment.  Km is a measure of substrate affinity for the enzyme.  A lower Km corresponds to a higher affinity.  The presence of an uncompetitive inhibitor actually increases the affinity of the enzyme for the substrate.  This surprising fact can be understood through the binding equilibrium.  Since the inhibitor binds the E-S complex, the inhibitor decreases the concentration of the E-S complex.  By Le Chatlier's principle, equilibrium of the enzyme and substrate will shift to form more E-S complex.  Therefore, the enzyme demonstrates a higher affinity for the substrate eventhough this higher affinity does not lead to a higher Vmax. 

In a Lineweaver-Burk plot, uncompetitive inhibitors shift the line higher with a raised y-intercept.

Scheme 4 - uncompetitive inhibitor model

Graph 1: Michaelis-Menten Plot

Graph2: The Lineweaver-Burk Plot

APPARATUS

Equipments

Eppendorf tubes

Erlenmayer flask

Micropipette

Cuvettes

Test tube rack

pH Meter

UV-VIS Spectrophotometer

Chemicals

Sodium phosphate buffer 0.05 M; pH 7

Mushroom tyrosinase

L-Dopa 7.60 x 10-3

PROCEDURE

With neutral pH, 0.05 M Sodium Phosphate Buffer was prepared to measure concentration of tyrosinase. 10 mg tyrosine was added to 100 mL of the buffer to get a 280 nm absorbance.

Dopa and Cinnamic acid solutions were prepared by using the buffer.

The activity of tyrosinase and dopa was measured by measuring the dopachrome levels.

The frequency of the light was adjusted to 475 nm.

First four steps were prepared before the experiment.

Five test tubes were prepared. By keeping L-Dopa constant at 0.75 mL, the buffer amount was made respectively 2.225, 2.200, 2.145, 2.100, 2.050 mL and tyrosine was added respectively again 0.025, 0.050, 0.100, 0.200 mL to make solution to 3 mL total.

The absorbance in every 30 seconds intervalin 2 minutes was measured with UV-VIS spectrophotometer.

Another five test tubes were prepared. By keeping tyrosinase constant at 0.05 mL, amount of L-Dopa was changed respectively 0.05, 0.10, 0.20, 0.40, 0.50, 0.75 mL and the buffer was added 2.90, 2.85, 2.75, 2.55, 2.45, 2.20 mL to the tube to complete it to 3 mL.

The absorbance of the solution was measured at 2 minutes.

Five test tubes were prepared. By keeping tyrosinase constant at 0.05 mL and cinnamic acid constant at 0.040, L-Dopa amount was changed respectively 0.05, 0.10, 0.20, 0.40, 0.50, 0.75 and buffer was added to the tubes respectively 2.55, 2.50, 2.40, 2.20, 2.10, 1.85 to complete all tubes to 3 mL.

The absorbance of the solution was measured at 2 minutes.

RESULTS

L-Dopa

(mL)

Buffer

(mL)

Tyrosinase (mL)

Absorbance

at 280 nm

30th Sec.

60th

Sec.

90th

Sec.

120th

Sec.

150th

Sec.

0.75

2.225

0.025

0.043

0.055

0.059

0.066

0.074

0.75

2.200

0.050

0.060

0.084

0.110

0.132

0.156

0.75

2.145

0.100

0.107

0.161

0.212

0.261

0.309

0.75

2.100

0.150

0.116

0.184

0.248

0.307

0.368

0.75

2.050

0.200

0.137

0.212

0.293

0.370

0.443

L-Dopa

(mL)

Buffer

(mL)

Tyrosinase

(mL)

Absorbance

at 280 nm

at 2 minutes

0.05

2.90

0.05

0.018

0.10

2.85

0.05

0.132

0.20

2.75

0.05

0.065

0.40

2.55

0.05

0.090

0.50

2.45

0.05

0.083

0.75

2.20

0.05

0.037

Cinnamic acid (mL)

L-Dopa

(mL)

Buffer

(mL)

Tyrosinase

(mL)

Absorbance

at 280 nm

at 2 minutes

0.40

0.05

2.55

0.05

0.070

0.40

0.10

2.50

0.05

0.036

0.40

0.20

2.40

0.05

0.030

0.40

0.40

2.20

0.05

0.037

0.40

0.50

2.10

0.05

0.055

0.40

0.75

1.85

0.05

0.066

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