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Enzymes


In this laboratory you will be introduced to enzymes, the "molecular working horses" of living organisms which speed up the thousands of chemical reactions of which life depends on. You will perform experiments with crude enzyme solutions or with highly purified enzymes and study their characteristics. You will study the effects of different experimental parameters and factors, such as pH, temperature and salinity on the catalytic activity of enzymes, which will help you to understand that enzymes are very specific protein-made biological catalysts which speed up chemical reactions thousand-fold. In preparation for this lab carefully rehearse the text book sections about enzymes and also carefully study the introductory part about enzymes of this web page.

Laboratory Objectives

After completion of this lab you should:
    - be familiar with the terminology related to enzymes and enzymology, including active site,
      activation energy, transition state, substrate, product and inhibitor
    - know what enzymes are and how they work; have an understanding of the catalytic cycle
      of an enzyme
    - have a basic understanding of enzyme kinetics and be familiar with the terms: specific enzyme
      activity, KM value and vmax, as well as be able to use the Michaelis-Menton Equation to do
      basic calculations
    - know which factors influence the catalytic activity of enzymes and why
    - know the difference between competitive and non-competitive enzyme inhibitors and be
      able to name examples
    - be able to set up simple enzyme testing methods (assays) to monitoring enzyme activity
    - to monitor and record changes in enzyme activity in the presence or absence of known or
      unknown enzyme inhibitors with the help of a VIS spectrophotometer
    - be able to analyze your enzyme kinetics and determine the mode of  enzyme inhibition with the
      help of graphical presentation techniques

Introduction

    One of the characteristics of life is the presence of a multitude of chemical reactions occurring
      within cells which in its total is defined as
metabolism

•    The chemical reactions occurring in living cells happen very rapidly and very selectively because
      of the presence of unique cellular components called enzymes
      - biochemists assume that for every chemical reaction happening within a cell, there is a
        specific enzyme present which helps to speed it up

•    Enzymes are biological catalysts, means cell constituents which speed up (or catalyze)
      chemical reactions
million-fold
     - without enzymes most chemical reactions would occur much too slowly to maintain life
        functions of a cell

•    Enzymes catalyze a chemical reaction by lowering the so-called activation energy  (EA);
      enzymes do not determine the direction in which a chemical reaction will proceeds nor do
      they change the chemical equilibrium constant (k) of the catalyzed chemical reaction
      (see
Figure 1 below)
      - activation energy is the energy push (or input) required to start a chemical reaction

Figure 1: Enzymes & Lowering of Activation Energy

•    The majority of enzymes in living organisms are proteins; as any protein they are build of
      hundreds of amino acids that are stringed together via so-called peptide bonds; the resulting
      polypeptide chain forms a highly complex 3-dimensional  structure, which is of crucial
      for the formation of the functional topography, e.g. the active site, and of prime importance
      for proper enzyme function

In order to give you a feel for the amazing variety (and beauty) in structures of enzymes, we will look into following  web site:

            http://www.biochem.ucl.ac.uk/bsm/enzymes

which offers access to a longer and longer getting list of enzymes and their unraveled 3-dimensional structures; we will use this web site to find the 3D-images and further background info of our two lab enzymes catecholase and alkaline phosphatase

•    Without enzymes cells would need high amounts of heat energy to start/trigger these
      necessary and life-sustaining chemical processes
      - high quantities of heat, usually necessary to trigger chemical reactions, would certainly
        destroy most of the temperature-sensitive biological structures in the cell

"Heat is not an option for life to do chemistry, and life had to come up with another
  solution to make chemical reactions possible at ambient, life-supporting temperatures ... "


      - heat is one form of energy that can be used to form chemical bonds and to produce bigger
        molecules
      - heat speeds up molecular motion and thereby increases the frequency of molecular
        collision and chemical bond formation
      - heat can also be used to break chemical bonds which hold the atoms of a molecule
        together; this is especially the case at very high temperatures

·        Enzymes use active sites (instead of heat) as crucial part of their complex 3-dimensional structures  to increase the frequency of molecular collision and to form or cleave chemical bonds
- with the help of usually narrow, cleft-like active sites enzymes dramatically increase the
  frequency of interaction between atoms or molecules

•   Only certain molecules are able to have access to the active site and perfectly fit into the active site of an enzyme (Fischer's "lock and key model"); these molecules are called the substrate of an enzyme; upon binding of a substrate molecule into the active site of an enzyme a fitted enzyme-substrate complex forms (Koshland's "induced fit model")

•   Upon successful binding of a substrate S in the active site of an enzyme and formation of the enzyme-substrate (ES) complex, a so-called transition state molecule S* of the substrate forms which finally changes into a new molecule, the so-called product P of the enzymatic reaction

•   After completion of one catalytic cycle (see Figure 2) the product is released (= dissociates) from the active site of the structurally (conformational) unchanged enzyme; the enzyme is capable to take up a new substrate molecule S and begin a new catalytic cycle

Figure 2: The catalytic cycle of an enzyme

·        The catalytic cycle of enzymes is enormously fast and (depending on the enzyme) many hundred up to thousands of substrate molecules per second (!!) can be “handled” during the enzymatic catalytic turn over

•  The catalytic activity of almost all biological enzymes identified so far, is dependent on either co-factors or co-substrates
- co-factors are inorganic substances, mostly the ionized atoms of important chemical elements
  such as iron, copper, magnesium, manganese, nickel, selenium, etc.
- co-substrates are larger, associated or covalently attached organic molecules, such as NAD+,
  FAD, biotin, thiamin, etc.
- both, co-factors and co-substrates are usually found located within or in close vicinity of the
  active site

The enzymes polyphenol oxidase (PPO; catecholase), amylase (AM) and alkaline phosphatase (ALP), you will be working with in this lab require copper (PPO), calcium (AM) and magnesium/zinc (ALP) as co-factors

•   The catalytic activity of enzymes can be measured with the help of so-called enzyme assays,
     to which principles you will be introduced in this lab course

•   Enzyme activity measurement  is usually based on the monitoring of the disappearance of the
     substrate molecule or the appearance of the product molecule over time

•   Most enzyme assays and enzyme activity monitoring methods relay on:

1.      Color changes (= changes in light absorbance) of the substrate or product
     molecules after enzymatic catalysis (= colorimetric or spectrophotometric
      enzyme assays
        - you will use this enzyme activity monitoring principle

2.      Changes in light absorption of enzyme-coupled co-substrates (e.g. NAD+)
     due to the catalytic activity of the enzyme

3.      Release of light after reaction of products with luminescent or fluorescent tracer
     molecules (= luminescent or fluorescent enzyme assays)

•   The measured catalytic activity of enzymes or specific enzyme activity is usually calculated and
     shown as nano or micro- mole substrate catalyzed per minute per milli-gram enzyme (or protein)
     and annotated in a text or a graphical presentation as:

                                    Specific enzyme activity v in [nmole / min x mg]
 

    - nmole substrate used or product formed
- mg protein or enzyme in the reaction

•   To compare the catalytic activity and velocity of different enzymes, protein biochemists relay on the Michaelis constant KM (or KM value), an important number in enzymology which can be experimentally
determined by measuring the enzyme activity at different substrate concentrations cS

•   To retrieve the KM value for a given enzyme, the enzyme activity v is graphically plotted against
     the substrate concentration cS (see Figure 3 below)

Figure 3: Enzyme Activity & Michaelis-Menton Kinetics

•   For most enzyme a typical “saturation curve” is observed which can be mathematically
    described by the so-called Michaelis-Menten equation (see Figure 3 above)
    - the Lineweaver-Burk plot (which is described with the help of the Lineweaver-Burk equation)
      is the double-reciproke "linear depiction" of the Michealis-Menton kinetics of an enzyme
    - it is often used for relatively easy and graphical finding of the KM value and of the maximum
      velocity (activity) of an enzyme

•   The KM value of an enzyme describes the strength of an enzyme or enzyme affinity for its
    specific  substrate; the smaller the KM value of an enzyme the stronger the affinity (= "desire to
    interact with") of the enzyme towards its substrate; the KM value is described by following formula:

                                                            KM =  k2 + kcat / k1

            - since for most enzymes kcat is much smaller (or less) than k2, we can set KM
                
equal to kd, which is the dissociation constant for binding of the substrate S to the
              enzyme E

·        If two or more enzymes interact with other, i.e. to form multi-enzyme complexes, and influence
each others catalytic activity in a positive or negative manner we speak of
positive or negative
cooperativity
; measurement of enzyme activity under these circumstances with increasing
substrate concentrations leads to a curve different to the "classical" Michaelis-Menton kinetic
we looked up in the section above; enzymes with cooperativity show a typical
"sigmoid curve"
in a linear plot (see
Figure 4 below)
- the sigmoid curve is mathematically described with the "Hill Equation" (see Figure 4 below)

Figure 4: Enzymes & Cooperativity

•   In a biological environment, e.g. the cell interior, enzymes do not always operate at the same pace;
 enzyme activities rather change in response to changes in the environment and cells are able to
 purposely and rapidly change enzyme activities as an adaptive response to environmental changes

   The catalytic activity of enzymes is influenced and affected by many factors, most importantly
 physical and physiological/cellular factors

·        Physical factors known to affect enzyme activity are:

        1. Temperature
         2. Salt concentration
         3. pH (= proton concentration)
         4. Chemicals (of synthetic or of natural origin)
            
- chemicals which are able to block the catalytic activity of an enzyme are often referred
             to as inhibitors
           - depending on the mode of action of a inhibitor molecule, we speak of competitive inhibitors
             (see Figure 5a) or non-competitive inhibitors (see Figure 5b)
           - many identified enzyme inhibitors of human enzymes are long known poisons and toxins
             (see hyper-linked Table below)

 

In this lab you try to find out which of the above factors will affect our lab enzymes and in which way!

        Physiological/cellular factors known to affect enzyme activity are many-fold and the ones which
        play a major role in enzyme regulation are listed below:

            1. Binding of hormones and cellular factors
            2. Covalent attachment of small molecules
                - most importantly attachment of phosphate groups (= Phosphorylation)
            3. Binding of so-called allosteric activators or inhibitors
                (see Figure 6 below)

•    If a chemical or an allosteric regulator molecule influence the enzyme activity by competing with
      the natural substrate for the active site, we speak in the language of enzymology of a so-called
      competitive inhibitor (see Graphic 5a below)

Figure 5a: Competitive Enzyme Inhibition

 

•   If a chemical or an allosteric regulator molecule binds at an enzyme region outside the active site and prevents the catalytic activity of the enzyme towards its natural substrate,  the molecule is referred to as a non-competitive inhibitor (see Graphic 5b below)

Figure 5b: Non-competitive Enzyme Inhibition

•  If an activated enzyme A interacts with one or more inactive enzymes of the same class and (upon
binding) lead to its/their activation we speak in the language of enzymology of positive cooperativity;
many enzyme have unique binding sites outside their active sites for docking of regulatory molecules or
even other enzymes; binding of this so-called allosteric regulator can - depending on the enzyme -
lead to either activation or inhibition of this enzyme, by a process called allosteric activation or
allosteric inhibition
(see Figure 6 below)
- allosteric activation plays an enormously important role in natural (= physiological) enzyme
  regulation in living cells

Figure 6: Allosteric Enzyme Regulation

•   Today we know that intracellular enzyme regulation happens at different levels and can be caused
 by all three regulatory mechanisms

•   All these regulatory factors are known or suspected to lead to a transient change of the intricate
(and important) 3-dimernsional structure of their target enzymes, thereby influencing their
catalytic activity, and ultimately to a different functional status of a cell
 

2. Enzyme Activity Testing Methods (or Enzyme Assays)

•   In this lab you will learn some of the basic tests biochemists use to determine the catalytic
     activity of purified enzymes or enzymes in crude cell extracts

•  Accurate enzyme analysis and assaying usually requires the purification of an enzyme from its
    source, e.g. a cell or a tissue, which can be achieved by different methods, most commonly by
    by gel filtration and affinity chromatography

•   Once an enzyme has been successfully purified or isolated from its natural (usually cell)
     environment, a scientist can begin to
characterize the isolated enzyme regarding its distinctive
    
enzyme properties such as:

    1. Type of Substrate(s)
2. Enzyme activity
3. Requirement of co-substrates and/or co-factors
4. Susceptibility towards
natural or synthetic regulators and/or inhibitor

 •    To achieve this, scientists apply fast and accurate enzyme testing systems (or
      
enzyme assays) to learn more about the enzyme’s properties

•    A “good” assay  has to minimize the possibility that the enzyme is denatured or degraded during
     the testing and that all known co-factors and co-substrates of the enzymes are included in the
     assay buffer; a good enzyme assay also is preformed at the optimum temperature, pH and salt
     conditions of the enzyme

•   Many different assay strategies have been developed to characterize enzymes, but assays where
    either the
decrease of the substrate or the increase of the product are monitored with different
    detection devices are the most common ones (for an overview: see
Figure 7 below)
    - enzyme activity can be monitored based on changes in:
            1. the spectral quality (e.g. absorption) of the substrate or product
                =
Colorimetric or UV/VIS-Spectrophotometric Assay
            2. the fluorescent or luminescent (light-emitting) properties of the substrate or product
                =
Fluorescent or Luminescent Assay
            3. radioactive properties of the (radio-labeled) substrate or product
                =
Radioisotopic Assay

 Figure 7: Different Enzyme Assay Methods & Strategies

•   Ideally the substrate changes color (= shift in it’s absorption spectrum) upon interaction with the
    enzyme, a property which can be easily monitored with the help of a
spectrophotometer
    (for function of a spectrophotometer: see separate Bio210A lab
section on this website)
    - e.g. TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine) changes color after oxidation by
      COX-2 enzyme
    - e.g. catechol (the substrate molecule you will be using in this lab) is converted into the
      brown-colored product benzoquinone by the enzyme Polyphenoloxidase (PPO)
    - e.g. BCIP/NBT tetrazolium salts are converted into colored diazo-compounds by the
      enzyme alkaline phosphatase (ALP)

You will learn how to operate and use a spectrophotometer to measure enzyme activity in this lab!

•  More complicated enzyme assays take advantage of the spectral changes of important (known)
co-substrates
of enzymes, such as NAD+ and NADP+, which can be sensitively monitored during
enzyme catalysis
- the reduced forms of NAD+ and NADP+ emit fluorescent light with a wavelength of 445 nm
  when excited at 340 nm which can be detected with a
UV or fluorescence spectrophotometer
- e.g. assaying the enzymatic conversion of lactate to pyruvate by the enzyme lactate dehydrogenase
  (LDH)
 is done in this way

If the lab time allows it and the department is in possession of a (usually expensive) fluorescence spectrophotometer or plate reader you will also learn how to operate and use this important lab equipment to measure enzyme activity in this lab!

•  The intrinsic fluorescence and UV-absorption properties of NAD+ and NADP+ are often exploited
in biochemistry labs in so-called
coupled enzyme assays, i.e. assays were there are usually two
or more enzymes in the assay test tube to detect for the presence of a certain molecule, e.g. ATP or
reduced glutathione (GSH) in a collected sample; in these assays the known specificity of the enzymes used is exploited to monitor changes in the assay signal in the presence of the molecule under investigation
(see Figure 8 below)
; coupled enzyme assays are commonly used for:

                1. ATP quantitation
            2. Measurement of Glutathione peroxidase activity

Figure 8: Coupled Enzyme Assays

•  Today, most enzyme assays relay on artificially labeled substrates which are offered to the enzyme
to sensitively trace their catalytic activity; the most common labels used in enzymology are:

1.      Chromogenic (= colored) substrate

- e.g. the elastin-labeling TPPS
- e.g. 2-naphtylamide which is converted to a colored azo-dye in the presence of diazonium
   salts
- 5-bromo-4-chloro-3-indolyl-b –galactopyranoside (X-Gal), a widely used artificial substrate
  in Molecular Cell Biology labs to monitor gene activation, is cleaved by the enzyme
   beta-galactosidase into a blue-colored compound

2.      Luminescent (= light-emitting) substrates or labels

    - D-luciferin cleavage by the fire-fly enzyme luciferase creates light
- Lucigenin or luminol molecules generate light after oxidation of hydrogen peroxide and in
  the presence of a unique class of enzyme called  peroxidases
- 1,2 dioxetanes (CDP-Star, etc.) are substrates of alkaline phosphatases and emit light after cleavage

3.      Fluorescent labels

    - fluorescent labels are (substrate-) attached molecules which emit light with a defined
   wavelength after (often laser-dependent) excitation
- prominent examples of fluorescent label molecules are:
            e.
g. BODIPY protein label
            e.g. Fluorescein
            e.g. Rhodamine
            e.g. Cy3

4.      Radio-labels

    - radio-labels are molecules which are radio-active due to the incorporation of a
  specific radio-activity-emitting isotope
- prominent examples of radio-isotopes widely used in enzymology for
  labeling of substrate molecules are:
           
35S (= radio-active sulfur for labeled of e.g. cysteine or methionine)
            14C (= radio-active carbon for labeled of e.g. glucose or fatty acids
            32P (= radio-active phosphorus for incorporation into phosphate or ATP)

     In this lab however you will conduct relatively simple colorimetric enzyme assays to measure the
 enzyme activities of one or two lab enzymes, e.g. PPO and ALP, under different experimental test
 condition as described in the "Procedures" part below; in both assays you will measure the formation
 of a colored enzyme (end) product as a measure for the enzyme activity in your test tubes

 

The files for the lab Procedures & Summary Questions can be retrieved by clicking
 on the "Interactive Buttons" below.

Procedure-CAT        Procedure-ALP        SummaryQ 

Important: Make sure that you bring a printed out version of the lab "Procedure" sheets (as
announced by your instructor) and of the
"Summary Questions" sheet with you to the
scheduled lab meeting!
(Both completed sheets are mandatory part of the weekly lab report!)