UNIVERSITY OF WESTERN SYDNEY - HAWKESBURY Enzyme Kinetics of Tyrosinase [Type the document subtitle]

Subject: Biochemistry 2.1

Aim: Using the enzyme Tyrosinase, (1) examine the relationship between enzyme co ncentration and reaction rate (2) Determine KM and Vmax values for Tyrosinase by varying the L-Dopa concentration, and (3) determine the mode of action of two T yrosinase inhibitors. Introduction: Enzymes serve as catalysts for biochemical reactions in living org anisms. They direct and regulate thousands of reactions providing for energy tra nsformation, synthesis and metabolic degradation. These catalysts speed up the forward and reverse reactions proportionately so th at, although the magnitude of the rate constants of the forward and reverse reac tions is increased, the ratio of the rate constants remains the same in the pres ence or absence of the enzyme. Enzymes increase reaction rates by decreasing the amount of energy required to form a complex of reactants (substrates) that is c apable of producing reaction products. The complex that forms when substrate (S) and enzyme (E) combine is called the e nzyme substrate (ES) complex. Reaction products arise when the ES complex breaks down, releasing free enzyme. Between the binding of substrate to enzyme, and the reappearance of free enzyme and product, a series of complex events must take place. An ES complex is formed which passes to a transition state (ES*) and then advances to an enzyme product (EP) that finally disassociates to a product and free enzyme. E + S ES ES* EP - E+ P

The kinetics of this type of reactions were first characterised by biochemists M ichaelis and Menten. Their basic equation to explain enzyme-catalysed reactions is: vo = Vmax [S]/ KM + [S] where: vo = initial velocity caused by substrate concentration [S] Vmax = maximum velocity KM = Michaelis constant. Biochemical Function Tyrosinase is the enzyme responsible for production of melanins and the darkenin g of skin. Persons with a genetic deficiency of tyrosinase have no skin pigmenta tion, i.e. albinism (Wilgram, 1967). The browning of apples, bananas and mushroo ms when exposed to oxygen is due to uncontrolled oxygenation and polymerisation of plant phenols catalysed by tyrosinase (Boyer, 2002) Activity: Tyrosinase is an oxygen-transferring enzyme that is tetramer (a macrom olecule produced by linking four identical monomers together) in form and contai

ns 4-gram atoms of copper per molecule (Jolley et al. 1974) and two binding site s for aromatic compounds including phenolic substrates. There is also a distinct ly different binding site for oxygen (Duckworth and Coleman 1970). Inactivation of the enzyme is associated with an increase in copper concentration (Kertesz et al. 1972). Besides using O2 to catalyse the dehydrogenation of catechols to orthoquinones a nd the hydroxylation of phenols to catechols, a peroxidase activity has been rep orted (Strothkamp and Mason, 1974). Optimum pH: 6.0 7.0 Inhibitors: Inhibitors are compounds that complex with copper. Also inhibited co mpetitively by benzoic acid with respect to catechol and by cyanide with respect to oxygen (www.worthington). Materials: [0.375 mL/100 mL ] Tyrosinase in 0.1 M phosphate buffer pH 7.0 in ice filled bucket. L-Dopa 15mM in buffer in foil covered tube. L-Dopa 1.5 mM (15 mM diluted with dH2o) Thiourea 20 mM Sodium Benzoate 4 mM in buffer 7 Eppendorf tubes Spectrophotometer and solution cuvettes Method: Part 1 7 Eppendorf tubes were placed into a tube rack with 0.25 mL L-Dopa added at a co ncentration of 15 mM into all 7 tubes. Following, for tube 1, 0.75 mL of phospha te buffer only was added. Tube 2, 0.65 mL phosphate buffer and 0.1 mL Tyrosinase were added, tube 3, 0.575 mL phosphate buffer and 0.175 mL Tyrosianse were adde d, tube 4, 0.5 mL phosphate buffer and 0.25 mL tyrosinase were added, tube 5, 0. 45 mL phosphate buffer and 0.3 mL tyrosinase were added, tube 6, 0.375 mL phosph ate buffer and 0.375 mL tyrosinase were added and for tube 7, 0.25 mL phosphate buffer and 0.5 mL tyrosinase were added. Tyrosinase was brought to room temperature before addition to tubes as this prov ides maximum reaction rate with the substrate. A spectrophotometer was zeroed, a nd, using tube 1 as the blank, 1 tube at a time was treated by the addition of t yrosinase in the above amounts. Tubes were mixed thoroughly by inverting with ca ps on, then solution placed in cuvette and analysed in spectrophotometer at an a bsorbance of 475 nm. Time was noted and absorbency values recorded every 15 seco nds for 2 minutes. Part 2: A table was provided to adjust buffer and enzyme quantities, so the final volume s of a second set of tubes (1 9) reached 1.0 mL Tube 1 was the blank, with 0.25 mL of 1.5 mM L-Dopa added and 0.75 mL of buffer. Tube 2 to 6 each had L-Dopa add ed at a concentration of 1.5mM at varying quantities 0.05 mL, 0.125 mL, 0.175 mL , 0.25 mL, 0.625mL respectively. Tubes 7, 8 and 9 had L-Dopa added at a concent ration of 15 mM and amounts of 0.125, 0.175 and 0.25 mL respectively. Final L-Dopa concentrations for tubes 1-9 were 0.0, 0.075, 0.188, 0.263, 0.375, 0.934, 1.88, and 2.63, 3.75 mM respectively. The spectrophotometer was zeroed using tube 1 (blank). Tubes 2-9 were filled wit h the required L-Dopa and buffer concentrations and quantities. Tyrosinase was added at intervals of 15 seconds for 2 minutes, placed in cuvette and analysed in the spectrophotometer with absorbances recorded. Part 3: In this part of the experiment we determined the mode of action of two Tyrosinas

e inhibitors, 1) Thiourea and 2) Sodium Benzoate. 1) Thiourea: Tube 1 was again the blank with 0.25 mL of 15 mM L-Dopa, 0.125 Thiourea and 0.625 buffer added. Tubes 2-5 had L-Dopa added at a concentration of 1.5 mM and amount 0.05, 0.125, 0.175 and 0.25 mL respectively. Tubes 6-9 had L-Dopa added at a concentration of 15.0 mM and amounts of 0.063, 0 .125, 0.175 and 0.25 mL respectively. Over all the tubes 1-9, Thiourea was added at 0.125 mL with buffer added to make up a total of 1.0 mL. Tyrosinase was added at 0.375 mL in tubes 2 9. 2) res In illuminating the mode of action for Sodium Benzoate, the same procedu were undertaken as above, but replacing Sodium Benzoate for Thiourea. For both inhibitors the spectrophotometer was zeroed with solution from tube 1. The enzyme Tyrosinase was added at room temperature, covered with parafilm and m ixed just prior to placing solution in cuvette. Each individual absorbance readi ng was recorded at intervals of 15 seconds over 2 minutes.

Results: The 4 tables below show the absorbance values recorded for each section of the l aboratory practical. Data Table 1 A475 Tube 2 15 s 0.029 30 s 0.047 45 s 0.057 60 s 0.069 75 s 0.079 90 s 0.088 105 s 0.097 120 s 0.109

Tube 3 0.033 0.053 0.071 0.090 0.109 0.126 0.142 0.157

Tube 4 0.057 0.080 0.106 0.130 0.153 0.175 0.196 0.219

Tube 5 0.061 0.088 0.121 0.148 0.178 0.203 0.230 0.256

Tube 6 0.073 0.111 0.148 0.185 0.220 0.248 0.253 0.314

Tube 7 0.106 0.156 0.206 0.254 0.297 0.336 0.366 0.380

Using Beers Law: [C] = A/ .l , A is entered to give [C] in Moles Litre-1min -1 which is then converted to mol/min. e.g. 0.253-0.111 = 0.142, 0.142/3600 = 3.94 x 10-5 M l-1min-1, which =(3.94 x 10-5) x 106 = 39.4 g/mL Conversion table 1 to determine Enzyme Concentration Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 V as A/min 0.041 0.071 V as mol/min 0.11 Enzyme Concentration g/mL 4 7 0.094 0.018 10 0.111 0.026 12 0.133 0.150 0.0318 0.041 15.8 21.6 0.056

Graph 1.

*note: Full size graphs in Appendix

Graph 2 Data Table 2 A475 Tube 2 15 s 0.022 30 s 0.028 45 s 0.031 60 s 0.035 75 s 0.039 90 s 0.042 105 s 0.045 120 s 0.048

Tube 3 0.033 0.043 0.053 0.061 0.069 0.077 0.084 0.090

Tube 4 0.030 0.041 0.053 0.063 0.074 0.083 0.092 0.100

Tube 5 0.040 0.055 0.069 0.084 0.097 0.111 0.123 0.135

Tube 6 0.055 0.081 0.106 0.130 0.155 0.178 0.199 0.222

Tube 7 0.108 0.146 0.180 0.212 0.243 0.273 0.300 0.323

Tube 8 0.094 0.133 0.166 0.202 0.236 0.266 0.297 0.323

Tube 9 0.121 0.159 0.198 0.230 0.266 0.299 0.330 0.359

Graph 3 Conversion Table 2 to determine Tube 2 Tube 3 Tube 4 V as A/min 0.013 0.031 0.035 V as mol/min 0.0037 0.0086 Substrate Concentration g/mL 0.075 0.188 0.263 Reaction Rates of Control. Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 0.059 0.097 0.0096 0.016 0.375 0.934 0.122 0.027 1.88 0.127 0.034 2.63 0.154 0.055 3.75 0.043

Data Table 3 A475 Tube 2 15 s -0.003 30 s -0.001 45 s 0.000 60 s 0.001 75 s 0.003 90 s 0.004 105 s 0.006 120 s 0.007

Tube 3 -0.002 0.000 0.001 0.002 0.004 0.005 0.007 0.008

Tube 4 0.000 0.001 0.003 0.006 0.008 0.010 0.011 0.013

Tube 5 0.010 0.013 0.016 0.019 0.022 0.025 0.026 0.030

Tube 6 0.024 0.033 0.042 0.050 0.057 0.064 0.070 0.076

Tube 7 0.021 0.029 0.037 0.044 0.052 0.058 0.065 0.071

Tube 8 0.018 0.026 0.035 0.043 0.051 0.059 0.066 0.073

Tube 9 0.023 0.047 0.036 0.048 0.053 0.058 0.069 0.077

Graph 4

Conversion Table 3 to determine Tube 2 Tube 3 Tube 4 V as A/min 0.0004 0.0004 0.008 V as mol/min 0.0011 0.0011

Reaction Rates of inhibitor Thiourea Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 0.012 0.028 0.032 0.032 0.02 0.0022 0.0033 0.0078 0.0089 0.0089 0.0056

Substrate Concentration g/mL 0.075 0.188 0.263

0.375

0.934

1.88

2.63

3.75

Data Table 4 A475 Tube 2 15 s 0.005 30 s 0.07 45 s 0.009 60 s 0.010 75 s 0.011 90 s 0.011 105 s 0.012 120 s 0.013 Graph 5

Tube 3 0.010 0.014 0.018 0.021 0.023 0.027 0.030 0.037

Tube 4 0.027 0.028 0.028 0.028 0.028 0.028 0.028 0.029

Tube 5 0.029 0.033 0.036 0.040 0.044 0.048 0.052 0.056

Tube 6 0.049 0.059 0.067 0.076 0.084 0.092 0.103 0.110

Tube 7 0.050 0.064 0.080 0.094 0.108 0.123 0.138 0.152

Tube 8 0.054 0.070 0.085 0.102 0.118 0.133 0.149 0.166

Tube 9 0.052 0.070 0.085 0.102 0.119 0.137 0.154 0.171

Conversion Table 4 to determine Tube 2 Tube 3 Tube 4 V as A/min 0.004 0.013 0.004 V as mol/min 0.0013 0.0028 Substrate Concentration g/mL 0.075 0.188 0.263

Reaction Rates of inhibitor Sodium Benzoate Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 0.016 0.032 0.058 0.064 0.0011 0.0044 0.0089 0.016 0.375 0.934 1.88 2.63 0.069 0.018 3.75 0.019

Below are the values given by SPSS computer programme for Km and Vmax using 3 di fferent plotting methods: Control Method Km Vmax Michaelis-Menten 0.925 0.139 0.0511 0.0028 Lineweaver-Burke 0.9 0.3662 0.161 0.0641 Hanes-Woolf 1.0 0.103 0.0524 0.0026 Thiourea Michaelis-Menten Lineweaver-Burke Hanes-Woolf 0.436 Sodium Benzoate Michaelis-Menten Lineweaver-Burke Hanes-Woolf 5.0 Method 2.3535 0.7251 0.0328 0.6295 0.6778 0.0094 4.4 0.0493 0.0408 Km 0.0051 0.0097 Vmax Method Km Vmax 0.5693 0.3525 0.00959 0.0019 0.4250 0.0.2498 0.0063 0.0034 0.349 0.0077 0.0015

Graph 6

Discussion: Enzyme inhibitors fall into two classes. There are those which cause irreversibl e inactivation of enzymes, and those in which their inhibitory effects can be re versed. Inhibitors of the first type usually cause an inactivating, covalent mod ification of the enzyme structure. (An example of this type of inhibitor is cyanide. By covalently binding mitochon drial cytochrome oxidase, it inhibits all the reactions associated with electron transport.) The kinetic effect of irreversible inhibitors is to decrease the concentration o f active enzyme, thereby decreasing the maximum possible concentration of ES com plex. Reversible inhibitors can be divided into main types, Competitive inhibitors and non-competitive inhibitors. There is also a type called uncompetitive inhibitor s but they are not often encountered. The common identifier of reversible inhibitors is that when the inhibitor concen tration drops, enzyme activity is regenerated. They usually bind to enzymes by n on-covalent forces with the inhibitor maintaining a reversible equilibrium with the enzyme. Competitive inhibitors have two means of reversal (1) decreasing the inhibitor c oncentration and (2) Raising the concentration of substrate (S) while holding th e concentration of the inhibitor constant as the substrate and competitive inhib itors both bind at the same site they compete with each other for binding, allow ing a second way of inhibition reversal. High concentrations of substrate can displace nearly all competitive inhibitor b ound to active sites. Thus, Vmax should be unchanged by competitive inhibitors. This trait of competitive inhibitors is reflected in the identical vertical-axis intercepts of Lineweaver-Burk plots, with and without inhibitor. Since attaining Vmax requires appreciably higher substrate concentrations in the presence of competitive inhibitor, Km (the substrate concentration at half maxi mal velocity) is also higher, as demonstrated by the differing negative intercep ts on the horizontal axis in panel B. Similarly, panel C illustrates that non-competitive inhibitors appear to have no effect on the intercept of the negative line implying that non-competitive inhi bitors have no effect on Km of the enzymes they inhibit. Since non-competitive i nhibitors do not interfere in the equilibration of enzyme, substrate and ES comp lexes, the Km s of Michaelis-Menten type enzymes are not expected to be affected by non-competitive inhibitors. However, because Enzyme Substrate complexes that contain inhibitor (ESI) are inc apable of progressing to reaction products, the effect of a non-competitive inhi bitor is to reduce the concentration of ES complexes that can advance to product . Since Vmax = k2 [Etotal], and the concentration of competent Etotal is diminis hed by the amount of ESI formed, non-competitive inhibitors are expected to decr

ease Vmax, as illustrated by the ordinate intercepts in panel C. A corresponding analysis of uncompetitive inhibition leads to the expectation th at these inhibitors should change the apparent values of Km as well as Vmax. Cha nging both constants leads to double reciprocal plots, in which intercepts on th e vertical and horizontal axis are proportionately changed; this leads to the pr oduction of parallel lines in inhibited and uninhibited reactions. Lineweaver-Burk Plots of Inhibited Enzymes http://web.indstate.edu

Inhibitor Type Binding Site on Enzyme Kinetic effect Competitive Inhibitor Specifically at the catalytic site, where it competes wi th substrate for binding in a dynamic equilibrium- like process. Inhibition is r eversible by substrate. Vmax is unchanged; Km, as defined by [S] required for 1/ 2 maximal activity, is increased. Non-competitive Inhibitor Binds E or ES complex other than at the catalyti c site. Substrate binding unaltered, but ESI complex cannot form products. Inhib ition cannot be reversed by substrate. Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration. Uncompetitive Inhibitor Binds only to ES complexes at locations other than the c atalytic site. Substrate binding modifies enzyme structure, making inhibitor- bi nding site available. Inhibition cannot be reversed by substrate. Apparent Vmax decreased; Km, as defined by [S] required for 1/2 maximal activity, is dec reased. http://web.indstate.edu

The standard curve of V vs. [E] corresponds with V being directly proportional t o E as long as S is being maintained at a high level. The graph representing V vs. [S] corresponds with that demonstrated in the lab, with Michaelin-Menten properties of a Saturation curve. There was only three err ant values that could not be fitted to the curved lines. The final data point of thiourea was lowered suggesting a error in sample preparation or recording valu es.

Conclusion: Thiourea demonstrated it is a non-competitive inhibitor. This is due to the fact that Km values are similar but Vmax values have decreased. This corresponds wit h the fact that non-competitive inhibitors bind to a place other than the active site, so preventing the maximum rate of reaction ever being reached. Sodium Benzoate showed that it did not respond as anticipated and recorded the h ighest Km value. This suggests that data was incorrectly interpreted or transpos ed. Non-competitive inhibitors are chosen for use in situations where the role of en zymes needs to be affected. This is achieved by selection of those with an activ e site different in shape to that of the substrate, so that they can no longer b ind. It is possible to predict which compounds will behave in this way by examin ing their chemical structure and determining whether they have a form similar to that of the substrate. Competitive inhibitors would be chosen where the rate o f the reaction needed to be slowed down, without affecting the overall outcome o f Vmax.

Examples of some non-competitive inhibitors are sodium azide, sodium citrate. Ex amples of some competitive inhibitors are L-phenylanaline, tryptophan and hydroxy quinoline. The results in part 1 were substantiated by the close relationship of the data p oints on the standard curve showing a direct relationship between [E] and Veloci ty.