Enzymes

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

Oxidoreductases

• 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

Transferases

• 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.

Hydrolases

• 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)

Lyases

• 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

Isomerases

• 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

Ligases

• 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 (v_max), 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 v_max, then K_m = [S]
• Michaelis Constant, K_m: 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 K_m has the lower affinity for its substrate since it requires a higher substrate concentration to be half-saturated
    • If [S] is below K_m, then changes in substrate concentration will greatly affect the concentration rate.
• V_max: Represents the maximum enzyme velocity and is measured in moles of enzymes per second
  • • • • 
• K_cat: measures the number of substrate molecules converted to product, per enzyme molecule per second.
  • At low substrate concentrations, K_m >>> [S], the Michaelis-Menton equation can be simplified to:
    • • • 
• Catalytic Efficiency: ratio of k_cat/K_m indicates the efficiency of the enzyme.

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/K_M
• Y-intercept is equal to 1/v_max

Pro bug catcher at the English Wikipedia, CC BY-SA 3.0, via Wikimedia Commons

Cooperativity

• 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

Temperature

• 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

• 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

Salinity

• 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 v_max
• Increases the value of K_m 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 v_max because there is less enzyme available to react
• Does not alter the value of K_M 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 K_M
  • If inhibitor preferentially binds to the enzyme, the K_M value is increased (lower affinity)
  • If the inhibitor binds to the enzyme-substrate complex, K_M value is lowered
• v_max 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 v_max and K_M

Lineweaver-Burk Plots for Inhibition

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

Zymogens

• 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)