Enzymes as Biological Catalysts

  • Enzymes are biological catalysts. A catalyst does not impact the thermodynamics of a biological reaction, only help the reaction proceed at a faster rate.

Enzymes Classifications

  • The molecule upon which an enzyme acts are called substrates
    • Enzyme specificity: a given enzyme will only catalyze a single reaction or a class of reactions with these substrates
  • Most enzymes have their named ending in the suffix ase

  • Catalyze redox reactions, usually with the help of cofactors to aid in electron carrying.
    • Electron donor is known as the reductant
    • Electron acceptor is known as the oxidant
  • Enzymes with dehydrogenase or reductase
  • Oxidase: enzymes with oxygen as their final electron acceptor

  • Catalyze the movement of a functional group from one molecule to another
  • Will be named with transferases in the name
  • Kinases are also a part of this group
    • Catalyze the transfer of a phosphate group, generally from ATP to another molecule.

  • Catalyzes the breaking of a compound into two molecules using the addition of water
  • Are named only after their substrate
    • E.g. – phosphatase cleaves a phosphate group from another molecule
    • Peptidases (proteins), nucleases (nucleic acid), lipases (lipids)

  • Catalyze the cleavage of a single molecule into two products
  • Do not require water and do not act as oxidoreductases
  • Reverse reaction can also usually be catalyzed by lyase (two molecules synthesize one)
    • Known as synthases

  • Catalyze the rearrangement of bonds within a molecule
  • Can also be classified as oxidoreductases, transferases or lyases sometimes
  • Catalyze reactions between stereoisomers and constitutional isomers

  • Catalyze addition or synthesis reactions, generally between large similar molecules and often require ATP.
  • Synthesis with smaller molecules is usually accomplished by lyases
  • Most likely to be encountered in nucleic acid synthesis and repair

Impact on Activation Energy
  • Endergonic reaction: requires energy input (DG>0)
  • Exergonic Reactions: energy is given off (DG<0)
  • Catalysts exert their effect by lowering the activation energy of a reaction.
    • Make it easier for the substrate to reach the transition state

Mechanism of Enzyme Activity

Enzyme-Substrate Binding
  • Molecule upon which an enzyme acts is called the substrate. Together the two are known as an enzyme-substrate complex.
  • Active Site: location within the enzyme where the substrate is held during the chemical reaction.
    • Assumes a defined spatial arrangement in the enzyme-substrate complex and this dictates the specificity of an enzyme for a molecule or group
  • Two competing theories explain the interaction between enzymes and substrates.

Lock and Key theory
  • Suggests that the enzymes active site (lock) is already in the appropriate conformation for the substrate (key) to bind.

Induced Fit Model
  • More scientifically accepted theory
  • Substrate induces a change in the shape of the enzyme
    • Interaction requires energy and is thus endergonic.
  • Once the substrate releases, the enzyme returns to its original state in an exergonic reaction.

Cofactors and Coenzymes

  • Cofactors or coenzymes are non-protein molecules which are sometimes required for enzymes to be effective.
  • Tend to be small in size so that they can bind to active sites of the enzyme
  • Usually carry a charge through ionization, protonation, or deprotonation.
  • Kept at low concentration so that they can be recruited when needed.
  • Apoenzymes: enzymes without their cofactor, while those with them are called holoenzymes.
  • Prosthetic Groups: tightly bound cofactors or coenzymes that are necessary for
  • enzyme function.
  • Cofactors are generally inorganic molecules or metal ions, and are often ingested
  • as dietary minerals.
  • Coenzymes are small organic groups, usually are a vitamin or derivatives of
  • vitamins (NAD+, FAD, coenzyme A)
    • Water-soluable vitamins: B complex vitamins, Vitamin C (ascorbic acid)
      • Must be replenished since they are easily excreted
    • Fat-Soluble vitamins: A, D, E and K
      • Regulated by partition coefficients which quantify the ability of a molecule to dissolve in a polar vs nonpolar environment.

Enzyme Kinetics

Kinetics of Monomeric Enzymes
  • Concentration of the substrate [S] and the enzyme [E] affects how quickly a reaction will occur.
    • Saturation: enzyme is working at a maximum velocity (vmax), and occurs when all active sites available are attached to a substrate.
      • Only way to increase rate is by increasing the enzyme concentration.

Michaelis-Menten Equation
  • Describes how the rate of reaction, v, depends on the concentration of both the enzyme [E] and the substrate [S], which forms product [P].
  • Concentration of enzyme is always kept constant
    • Velocity of the enzyme can be related to the substrate concentration:
      • When this equation is equal to half of vmax, then Km = [S]
  • Michaelis Constant, Km: is the substrate concentration at which half of the enzymes active sites are full
    • Used as a measure to compare enzymes since it measures the affinity of the enzyme to its substrate.
      • The one with the higher Km has the lower affinity for its substrate since it requires a higher substrate concentration to be half-saturated
      • If [S] is below Km, then changes in substrate concentration will greatly affect the concentration rate.
  • Vmax: Represents the maximum enzyme velocity and is measured in moles of enzymes per second
  • Kcat: measures the number of substrate molecules converted to product, per enzyme molecule per second.
    • At low substrate concentrations, Km >>> [S], the Michaelis-Menton equation can be simplified to:
  • Catalytic Efficiency: ratio of kcat/Km indicates the efficiency of the enzyme.
Michaelis Menten curve 2
Saturation curve for an enzyme reaction showing the relation between the substrate concentration and reaction rate.
Thomas Shafee, CC BY 4.0, via Wikimedia Commons

Lineweaver-Burk Plots
  • Double reciprocal graph of the M-M equation. This graph yields a straight line
  • Only real data is to the left of the y-axis (QUAD 1)
  • X-intercept is equal to -1/KM
  • Y-intercept is equal to 1/vmax

  • Certain enzymes do not show classic hyperbola shape when M-M equation is graphed, instead show S-shaped sigmoidal due to cooperativity among substrate binding sites
  • Cooperative enzymes have multiple subunits and multiple active sites
    • These subunits and enzymes may exist in one of two states
      • Low-affinity Tense state (T)
      • High-affinity relaxed state (R)
    • Binding of substrate encourages the transition of other subunits from the T state to the R state, which increases the likelihood of substrate binding to other subunits.
    • Conversely, loss of a substrate can encourage other subunits to move from R state to T state.
  • Often shown in regulatory enzymes inn pathways
  • Quantified using Hill’s Coefficient
    • Hill’s Coefficient > 1: positively cooperative binding
      • After one ligand is bound the affinity of the enzyme for further ligands increases
    • Hill’s Coefficient ><1: negatively cooperative binding
      • After one ligand is bound the affinity of the enzyme for further ligands decreases
    • Hill’s Coefficient = 1: enzyme does not exhibit cooperative binding

Effects of Local Conditions on Enzyme Activity

  • Enzyme activity, Enzyme velocity, and enzyme rate are used interchangeably

  • Enzyme-catalyzed reactions tend to double in velocity for every 10 degree increase in temperature until an optimum temperature is reached (37°C/98.6°F/310 K)
    • After optimum temperature is reached, activity falls of sharply if temp is increased
  • Some enzymes are able to regain their function once cooled down.

  • pH affects the ionization of the active sites
  • A change in pH can also cause the denaturation of enzymes.
  • Optimal pH is 7.4. Acidemia is when blood pH is less than 7.35
    • Exceptions to this optimal level occur in the digestive tract
      • Pepsin (stomach) works at a pH of 2
      • Pancreatic Enzymes work best in the small intestine at a pH of 8.5

  • Altering the concentration of salt can change enzyme activity in vitro
  • Increasing levels of salt can disrupt hydrogen and ionic bonds which would cause a partial change in the conformation of the enzyme

Regulation of Enzyme Activity

Feedback Regulation
  • Feedback regulation: Enzymes are often subject to regulation by products further down a given metabolic pathway
  • Feedforward regulation: enzymes regulated by intermediates that precede the enzyme in the pathway. Less common
  • Negative Feedback/Feedback inhibition: once we have enough of a given product, the pathway that creates the product should be turned off
    • Most common
    • Product may bind to the active site of an enzyme to competitively inhibit the enzymes and make them unavailable for use

Reverse Inhibition
  • Four types: competitive, noncompetitive, mixed, and uncompetitive

Competitive Inhibition
  • Involves the occupancy of the active site since substrates cannot access the enzymatic binding sites if there is an inhibitor in the way.
  • Can be overcome by adding more substrate to increase the chances of it displacing the inhibitor.
  • Adding a competitive inhibitor does not alter the value of vmax
  • Increases the value of Km since the substrate concentration has to be higher to reach half of the maximum velocity

Noncompetitive Inhibition
  • These inhibitors bind to allosteric sites instead of active sites
  • Allosteric Sites: non-catalytic regions of the enzyme that bind regulators
  • Inhibition cannot be overcome by additional substrate since the two are not competitive.
  • Bind equally well to the enzyme or the enzyme-substrate complex
  • Decreases the measured value of vmax because there is less enzyme available to react
  • Does not alter the value of KM since the affinity of unaltered enzymes stays unchanged.

Mixed Inhibition
  • Inhibitor can bind to either the enzyme or the enzyme-substrate complex, but the affinity for each is different
  • Bind at allosteric site
  • Alters the experimental value of KM
    • If inhibitor preferentially binds to the enzyme, the KM value is increased (lower affinity)
    • If the inhibitor binds to the enzyme-substrate complex, KM value is lowered
  • vmax decreases regardless of the affinity

Uncompetitive Inhibitors
  • Bind only to the enzyme-substrate complex and essentially lock the substrate in the enzyme
  • Can be defined as increasing affinity between the enzyme and substrate
  • Must bind to an allosteric site
    • The enzyme-substrate complex induces a conformational change that allows the uncompetitive inhibitor to bind
  • Lowers vmax and KM

Lineweaver-Burk Plots for Inhibition
Enzyme Inhibition lineweaver-burk plots
Bizz1111, CC0, via Wikimedia Commons

Irreversible Inhibition
  • The active site is made unavailable for a prolonged period of time or is permanently altered.
  • E.g. – Aspirin and other pain killing drugs are used to permanently disrupt the functioning of enzymes that help on creating pain-modulating products.

Regulated Enzymes

Allosteric Enzymes
  • Have multiple sites: one active site and at least one allosteric site
  • Allosteric Sites: regulate the availability of active sites
  • Allosteric Enzymes: alternate between an active and inactive form
    • Inactive form: cannot carry out the enzymatic reaction
  • Molecules that bind to the allosteric site can be either allosteric inhibitors or allosteric activators.
    • Binding of either causes a conformational change in the protein
      • An activator will result in a shift that makes the active site more available
      • Inhibitor will make active sites less available

Covalently Modified Enzymes
  • Enzymes can be activated or deactivated by phosphorylation or dephosphorylation
    • Cannot determine whether it is activated or deactivated with experimentation
  • Glycosylation is the covalent attachment of sugar moieties
    • Can tag an enzyme for transport within the cell or can modify protein activity and selectivity

  • Inactive form of potentially dangerous enzymes
  • Contain a regulatory domain and a catalytic (active) domain.
    • Regulatory domain must either be altered or removed to expose the active site
  • Have the suffix –ogen usually
  • E.g. – trypsin has a zymogen form trypsinogen; Apoptotic enzyme (caspases)