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Enzyme inhibitors (DOC)

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					                                       Enzyme inhibitors

Background

The activity of many enzymes can be decreased by exposure to a variety of compounds. These
compounds interact with the enzymes to lower their activity or inhibit them. The use of enzyme
inhibitors yields valuable information concerning the mechanism of enzyme action, possible
isolation methods using affinity chromatography, active site modifications and structural analysis,
and in designing new types of drugs.

Identifying and classifying inhibitors into various categories can be accomplished using enzyme
kinetics and graphical methods of analysis. Each inhibitor affects the Km, Vmax or both the Km and
Vmax. The manner and degree of this effect can be observed by looking at changes in Lineweaver-
Burk, Hanes, Eadie-Hofstee, Direct Linear, or Wilkinson plots. From the patterns of lines one can
classify a specific inhibitor and determine apparent Km and Vmax for the substrate. A Ki
(dissociation constant) can also be determined for the inhibitor.

Inhibitors can be classified in many ways. Irreversible inhibitors (Hg, Pb, Cd, nerve gases, etc.)
usually bind to a specific residue in the enzyme and block catalysis either by modifying the active
site of disturbing the conformation of the protein. These inhibitors are typically called poisons.

Competitive inhibitors (CI) bind to the free enzyme at the active site. They have a structure similar
enough to the substrate to become a substrate look alike. Because of their ability to fit into the
active site catalysis is blocked.

E + S         ES         E + P
E + I         EI
(E x I) / EI = Ki

From this relationship a new Michaelis-Menten equation can be written.


   v = Vmax x S
     ____________________

       Km ( 1 + I/KI ) + S


Measurement of v at various concentrations of S in the presence of a constant amount of inhibitor
yields a Lineweaver - Burk plot similar to that below. Competitive inhibitors usually change the
Km but not the Vm. Increasing the substrate concentration can also overcome inhibition by
competitive inhibitors.




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A direct linear plot for a competitive inhibitor gives a pattern similar to the one below.




Noncompetitive inhibitors can bind to free E or the ES complex. Usually this binding takes place at
                                                   2
some other site than the active site. Binding at this site does affect binding at the catalytic site. In
contrast to competitive inhibitors, increasing the substrate concentration cannot overcome this type
of inhibition. Noncompetitive inhibitors usually affect the Vmax, but some can affect both the Km
and Vmax.

E + S        ES     E + P
E + I        EI
ES + I       ESI
E x I) / (EI) = KIE and (ES x I) / ESI = KIS

Double reciprocal plots yield results similar to those seen below.




Uncompetitive inhibitors are relatively rare. This type of inhibitor combines only with the ES form
of the enzyme. Because of this both the Km and Vmax are changed.
                                                    3
E + S        ES    E + P
ES + I       ESI
(ES x I) / ESI = KI

Double reciprocal plots look similar to those below.




Based upon the patterns of inhibition, line patterns in graphs, a guess can be made as to what type of
inhibitor is affecting the enzyme. The plots above are "ideal" and over simplified. Similar patterns
can also be described by other phenomena associated with changes in enzyme kinetics.

The table below does, however, describe the qualitative changes in the slope and intercept for each
inhibitor.




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Tyrosinase inhibitors

Tyrosinase is a copper containing oxido-reductase that also contains free cysteine(s). Over the
years, many inhibitors have been found that inhibit the enzyme in some manner. Some of the
inhibitors are poisons while others act to reduce quinones back into phenols. Others can act as
copper chelators. Some are competitive and others are noncompetitive. In general, inhibitors fall
into two categories: those that affect the Cu and those that affect the phenolic binding site. Since
there is an oxygen binding site, inhibitors such as carbon mono/dioxide also inhibit tyrosinase. See
table below for a partial list of inhibitors. Many of the inhibitors in this table fall into one or more
of the suggested categories.

Cu chelators                    reducing agents                 competitive              noncompetitive

EDTA                            ascorbate                       hydroxamic acid       phenylhydrazine
diethyldithiocarbamate          mercaptoethanol                 cinnamic acid         anisaldehyde
phenylthiourea                  bisulfite                       p-coumaric acid       (2E)-alkenals
sodium azide                    dithiothreitol                  2,3-naphthalenediol
methimazole                     glutathione                     tropolone
tropolone                       cysteine                        mimosine
hydroxamic acid                 thioglycollate                  benzoic acid
kojic acid                                                      quercetin
maltol                                                          salicylhydroxamic acid
cyanide

Miscellaneous                                                   Quinone conjugators

polyvinylpyrrolidone (PVP)                                      methimazole
H 2O 2                                                          kojic acid
honey
borate
natural peptide/protein inhibitors
4-hexylresorcinol




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Experimental

A. Determination of a suitable inhibitor concentration

1. Use the standard assay conditions that were determined in the lab last week. Use an amount of
   enzyme that gives a slope (initial velocity) of approximately 75 o for each assay.
       enzyme + buffer 0.5 ml
       substrate/buffer    2.5 ml

2. Vary the amount of inhibitor added in each assay, but keep the substrate and enzyme
   concentrations constant as well as the total volume. For example,
       enzyme              0.1 ml
       substrate/buffer 2.5 ml
       inhibitor           0.4 ml

   Other inhibitors that can be tested are tropolone, salicylhydroxamic acid (SHAM), and
   4- hexylresorcinol. These inhibitors are effective in the micromolar range.

3. Determine the initial velocities from the linear portion of the curve. Plot v vs I. Choose a value
   of I that will decrease the I initial v by about 15% (see example below).
     sample          ml I       v

        1            0.01
        2            0.05
        3            0.1
        4            0.2
        5            0.3

B. Determination of the type of inhibition

1. Set up a series of 10-12 standard assays containing a variable concentration of substrate (see
   instructor). Keep the enzyme amount and the inhibitor amount constant in each assay. For
   example,
        enzyme              0.1 ml
        inhibitor          0.1 ml
        substrate/buffer 0.05 - 2.5 ml
        buffer             to 3 ml

2. Determine v from 0.5x the Km to 5x the Km in the presence of the inhibitor, using an I
   concentration that decreases v by approximately 15%.

3. Calculate v from the initial part of the curve or from the linear portion of the curve.

4. Do a Michaelis - Menten plot, Lineweaver - Burk plot, Hanes, Eadie, and Direct linear plot.
   Determine apparent Km and Vmax values for L - dopa or catechol in the presence of inhibitor.

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5. Compare these values to those obtained in the absence of an inhibitor.

6. Compare your data to other group data using different tissues or different types of mushrooms.


The following graphs and tables will be handed in with your report.

1. v vs E

2. v vs S ; plus or minus I

3. 1/v vs 1/S ; plus or minus I

4. v vs I

5. Table 1
                                     -I                             +I
                                Km        Vmax                 Km        Vmax

       Lineweaver-Burk

       Hanes-Woolf

       Eadie-Hofstee

       Wilkinson,
       (nonlinear, v vs S)




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