Lecture 6: Biochemical Reactions (Enzymatic Reactions)

Biochemical Reactions

• chemical reactions that take place in the cells of living organisms

• commonly organized in specific biological pathways

• must be enzyme-driven and exergonic

Enzymes

• biological catalysts that speed up biochemical reactions

• not consumed or generated, gets conserved

• decreases activation energy, but do not impact the Gibbs free energy

Reaction Rate

• kinetic control; how easy it is to overcome activation energy

• collision theory applies

• the amount of product formed or substrate consumed per unit of time

  • ratio of change in concentration to change in time

Enzyme-Driven

all biochemical reactions in the cell are catalyzed by specific enzymes

Exergonic

• always move forward, move in just one direction in metabolic pathway: energy flow

• either metabolically reversible or irreversible biological reactions

Reaction Direction

thermodynamic control, how to make Gibbs free energy more negative?

laws of thermodynamics apply

Structural Protein

keratin of hair and nails, collagen of bones

Enzymes or Catalytic Protein

hexokinase, pepsin

Hormonal Proteins

insulin, growth hormone

Transport Protein

hemoglobin, p-glycoproteins, serum albumin

Receptor Proteins

for hormones, viruses

Contractile Proteins

actin, myosin

Storage Protein

ovalbumin, glutelin

Genetic Proteins

nucleoproteins

Defense Proteins

snake venom, immunoglobulins

Naming Enzymes

• nomenclature of enzymes usually ends with -ase

• common names: trypsin, pepsin, chrymotrypsin

Protein-Type Enzymes

any pure proteins or conjugated proteins (e.g. the proteins containing metal ions) produced by living organisms and functioning as specialized biological catalysts for selective metabolic processes of an organism

Protein Enzyme Principles

same principles that apply to proteins e.g. enzymatic protein digestion, denaturation by heating (unfolding and refolding, pH EXCEPT RIBOZYMES

Ribozymes

biocatalysts made from RNA molecules e.g. peptidyl isomerase

Significance of the Enzymes in Biomedical Science

• proteins such as enzymes, receptors, (G protein-coupled receptors + nuclear hormone receptors), transport proteins, and immunoproteins are the most common biological targets of pharmacologically active drugs, toxic agents, and many hormones

• will follow the Law of Mass Action: M-M kinetics or Hill Kinetcs

Life

a quality that distinguishes matter reflected by biological processes, such as signaling and self-sustaining processes, from matter, which does not

Biological Processes

• life activities that an organism performs to survive

• made up of a group of biological reactions

Enzymes and Life

if one can control the enzymes, the biological reaction, biological process, and life can be consequently controlled or managed: the enzymes are one primary target of drugs/toxicants

Enzyme-Mediated Reaction

converting the substrates into products: E-S complex generated

Intermediate States

• less stable

• also called transition state

Activation Energy

• follows collision theory

• difference in energy level between the substrate and transition state

• reaction catalysts, including enzymes, lower activation energy

• the higher the activation energy, the lower the chemical reaction rate constant will be

• a reaction can only occur once it reaches the top of the activation energy barrier

Structure Features of Enzymes

• proteins

• do not change the chemical transformation of the substrates: similar to non-enzymatic reactions

  • catalyzing the same type of reactions: oxidation, reduction, hydrolysis

  • producing the same products

Enzyme Structure: Ligand Binding

ligand can be a substrate or a modifier

Active Site

• the surface of the enzyme where the substrates bind

• bind in precise orientation with other ancillary groups

• commonly polar amino acids are located here

• some regulatory molecules (modulators/modifiers) can also bind to

Allosteric Site

• the bindings sites other than active site that binds to regulatory molecules (modulators/modifiers) in the cellular environment

• may form weak non-covalent bonds with the molecules in a reverisble enzyme inhibition

• consequently, causing a change in the conformation of the enzyme that can translate to the active site, affecting the reaction rate of the enzyme

  • e.g. noncompetitive inhibitors on free enzyme

• substrates can’t bind

Thermodynamics

• deals with direction (spontaneousness) in which a process occurs but tells nothing about its rate: work, heat, energy, and matter states

• reaction direction

• stable systems

• Gibbs free energy

Reaction Kinetics

• branch of physical chemistry that is concerned with understanding the rates of chemical reactions

• laws of movement under forces

• reaction rate: how fast

• transition systems

• activation energy

Enzyme Kinetics

the mathematical description of the enzyme actions, especially the rates of an enzyme-driven chemical reaction

Law of Mass Action

the rate of a chemical reaction is proportional to the “active mass” (molar concentration) of the reactants that selectively bind to a protein including substrate-enzyme binding kinetics, substrate-transporter binding kinetics, ligand-receptor binding, and antigen-antibody binding

M-M Kinetics

• monosubunit enzymes: single polypeptide

• more specifically, the enzymes with only one active site

• special type of Hill kinetics

Hill Kinetics

followed by allosteric enzymes

Allosteric Enzymes

mostly multisubuit enzymes: enzymes with two and more active sites

Enzyme Activity

• the measure of the ability of an enzyme to catalyze a specific reaction, usually expressed as the rate of the reaction

• number of moles of substrate converted into product per unit time

• determines enzyme kinetics

• determined by performing the enzyme activity assays

Enzyme Activity Key Parameters

V_max, K_m, and slope (V_max/ K_m)

Enzyme Activity Assay

• substrates and detection methods e.g. alkaline phosphatase

• requirements: substrate(s), enzymes, reaction buffer, reaction incubator, detection methodology (commonly spectrophotometer)

Traditional Enzyme Kinetics

portion of curve of generated products vs reaction time illustrated by dashed line within limited recorded time

Reaction Velocity/Rate

• the change in amount/concentration of a reactant or product per unit time

• the slope the curved plot at different time point

• variable when the reaction moves on

• values of reaction rate vary, often go down with time

Initial Velocity

• V_i or V₀: the slope of the dotted line when t = 0

• instantaneous reaction rate at the start of the reaction

• most rapid rate in an enzyme-driven reaction

• an actual/real velocity that can’t be directly measured by can be calculated

Simplest Enzyme Reaction

substrates to products

Enzymes Simple Kinetic Mechanisms

• enzyme binds substrate

• enzyme transforms the substrate into product

• enzyme releases the product

Enzymes Kinetic Mechanism Process

• Enzyme + Substrate → Enzyme-substrate complex (ES)

• ES → Enzyme + Product

• net reaction: enzyme + substrate → enzyme + product

Two Forms of Enzymes

free enzyme (E), conjugated enzyme/enzyme-substrate (ES) mixture

Rate Constant (K)

• proportionalities between substrate concentration and reaction rate

• k = reaction rate[substrate]

  • k value depends on temperature and activation energy

K_f or K₁

forward rate constant for the formation of ES

K_r or K_-1

the reverse rate constant for the formation of E and S from ES

K_cat or K₂

catalytic rate constant that leads to product formation, or the constant for the breakdown of ES to E and P

Michaelis-Menten Equation

• V_i = initial reaction velocity

• V_max: maximal velocity

• [S]: substrate concentration

• K_m: steady state constant/Michaelis constant

Michealis-Menten Kinetics (Basic)

based on two step enzyme driven reaction: [Enzyme] total = [E] free + [ES]

Key Assumption for M-M Kinetics

• necessary to derive an equation from the model that relates initial velocity to substrate concentration

• the enzyme substrate complex is under steady state, or steady state conditions hold for almost all enzymes

  • d[ES]/dt = 0

  • k_r >> k_cat or k_-1 >> k₂

Steady State of Enzyme-Mediated Reaction

the concentration of enzyme-substrate complex [ES] is constant with time

Enzyme Conservation

• sum of total amount of all forms of enzyme, not altered before, during, and after the reaction

• however, the configuration/format of the enzymes can be altered: deprotonated, oxidixed, reduced

Michaelis-Menten Equation Breakdown

• relates initial velocity to substrate concentration: the initial rate/velocity of an enzyme-catalyzed reaction is dependent on the substrate concentration

• only applied to monomer enzyme: single polypeptide protein

M-M Kinetics System Application

• substrate-enzymes

• substrate-transporters: gluts

• pO2—oxygen saturation of myoglobin

• antigen-antibody

• ligand-receptors

Enzyme Kinetics Breakdown

• essentially enzyme-substrate binding kinetics: depending on ES or the level of substrates that is bound to active sites of enzyme

• we assume that once the substrate binds to enzyme, step 2 will automatically happen

• but, the binding of enzymes to substrate is the key step

• enzyme activity depends on how fast ES complex is formed

Michaelis-Menten Plot

• hyperbolic

• M-M equation predicts the generation speed/rate of the end production

• represents steady state with net flow of substrate to product

• Saturable due to limited amount of enzymes

K_m (Michaelis Constant), Graph

• determination of K_m value: the [substrate] at ½ V_max

• K_m of substrates at cellular concentration in most biological enzymes: µM

• different substrates of the same enzyme have a different K_m which can be used to differentiate between them

• lower K_m indicates higher/tighter binding affinity

• feature of a specific enzyme to a specific substrate at a specific condition such as pH, temperature, buffer system

K_m

• indicates bindng affinity of an enzyme to a specific substrate

• lower K_m means higher affinity, often means more substrate in active sites when given the same amount of substrates

V_max

• indicates binding capacity of an enzyme to a specific substrate

• higher V_max of a similar reaction means higher binding capacity

Slope (V_max/K_m)

• a measure of catalytic efficiency

• best value to represent the enzyme’s overall ability to convert substrate into product

• higher V_max/K_m ratio means higher catalytic efficiency

  • when K_m >> [S]; V_i = (V_max/K_m) x [S]

Binding Abundance

• reaction rate is proportional to binding abundance

• substrate x binding affinity of substrate to enzyme x binding capacity of the enzyme to substrate

Binding Capacity

indicted by number of active sites, proportional to [total enzyme]

Impact of Slope on Catalytic Efficiency

• the higher initial velocity, the faster substrates convert into products, the higher catalytic efficiency

• enzyme 1 always catalyzes the conversion of substrates into products faster than enzyme 2, higher catalytic efficiency

• indicated by that initial velocity is always higher in enzyme 1

Impact of Slope on Aleration of V_i

• when the same amount of substrate is provided, system 1 with a higher slope increases initial velocity faster than system 2 with a lower slope

• system 1 catalyzes the conversion from substrates to products faster than system 2: more efficient

Trans-membrane Proteins

transporters

Features of Membrane Transport Proteins

• allows specific molecules or class of molecules to cross the membrane

• ex. glucose transports

  • follow M-M kinetics when graphed

Double-Reciprocal or Lineweaver-Burk Plot

• M-M curve (hyperbolic) can be transformed into a straight line

  • plot = 1/v vs 1/[S]

• values of K_m and V_max can be read from the intersections of its axes

Regulation of Enzyme Activity

• alteration of expression levels of the enxyme protein

  • changing enzyme total leads to change in Vmax

• alteration of configuration/3D structure of the enzyme via modification

  • may change Km, Vmax, and or Vmax/Km

  • stimulation

Up-Regulation

increase in enzyme total leads to increase in Vmax

Down Regulation

decrease in enzyme total leads to decrease in Vmax

Inhibition

• most common in the biological system

• classification: based on the interaction between the inhibitor and enzyme

Activator

a molecule that will increase the function of a target protein: enzyme activity, transport function, etc

Inhibitor

a molecule that will decrease the function of a target protein

Reversible Inhibition

• forming non-covalent bonding

• an inhibitor that can bind to and dissociate from an enzyme until reaching equilibiurm

• bind to the active or allosteric sites of enzymes

• can bind to free and conjugated enzymes

• three main types: competitive, anti/un-competitive, mixed/noncompetitive

Reversible Inhibition Mechanisms

• free: EI produces E and I

  • K_i = [E][I] / [EI]

• conjugated: ESI produces ES and I

  • K_i = [ES][I] / [ESI]

Original Vmax or Km

Vmax or Km in the absence of inhibitor/activator

Apparent Vmax or Km

Vmax or Km in the presence of inhibitor/activator

Competitive Reversible Inhibition

• inhibitor binds to free/unconjugated enzyme only

• I and S competitively bind to same active site that has a chemical structure similar to the substrate

• M-M equation changes

• effective at low substrate concentrations because the substrate and inhibitor have a similar structure, the chance of either of them binding to the active site is the same

Competitive Reversible Inhibition M-M Equation Change

• no change in Vmax

• Km is increased

• the slope is decreased

Competitive Reversible Inhibition: Occupation

• more occupation of active sites by inhibitors indicates higher inhibitoin

• less occupation by inhibitor indicates less inhibition

Apparent Km: Km’

the Michaelis constant as observed under conditions that would hinder the determination of its true value e.g. the presence of a competitive inhibitor

Apparent Vmax (Vmax’)

• Vmax = Kcat [Enzyme] total

• no change of the total amount of the enzyme

• no change of Kcat

• Vmax’ = Vmax

Anticompetitive/Uncompetitive Reversible Inhibition

• the inhibitor binds to ES complex only: allosteric site

• do not structurally resemble substrate

• ESI intermediate forms

• at the point where substrate concentration reaches 0, no effect of the inhibitor

• inhibition is strongest when [S] reaches infinity

Noncompetitive/Mixed Reversible Inhibition

• the inhibitor binds to both free E and conjugated enzyme ES

  • inhibitors bind to the enzymes at the site other than the active site: allosteric site

• at low substrate concentration, inhibition resembles competitive

• at high substrate concentration, inhibition resembles anticompetitive

Double Reciprocal Plot of Reversible Enzyme Inhibition

• competitive: lines intersect on Y axis

• anticompetitive: lines are parallel

• noncompetitive: lines intersect on X-axis

Irreversible Enzyme Inhibition

• modifying agent/the inhibitor is covalently attached to the enzyme and cannot be removed

  • covalent bonds are much stronger than non-covalent bonds

  • causing enzyme with little or no activity left: kills enzyme

• enzyme activity can be restored only after synthesizing new enzymes

• common toxication mechanism of a toxicant/toxin

Organophosphates: Irreversible Enzyme Inhibitors

• nerve gas, diisopropylfluorophosphate (DIPF) and pesticide parathion

  • irreversible inhibitors of acetylcholine esterase lead to breakdown of acetylcholine

  • if the enzyme cannot work correctly, muscles go into uncontrolled contraction and death results: too much acetylcholine

Drug Examples (Aspirin): Irreversible Enzyme Inhibitors

cyclooxygenases (COX) inhibits prostaglandin and prostacyclin formation

Drug Examples Penicilin: Irreversible Enzyme Inhibitors

DD-transpeptidase disrupts the formation of bacterial cell walls

Drug Examples (Prilosec, Omeprazole): Irreversible Enzyme Inhibitors

• H+/K+—ATPase inhibits proton pump in the stomach

  • treatment of gastroesophageal reflux disease, gastric and duodenal ulceration, and gastritis

Reversible vs Irreversible?

• Enzyme activity/function: reversible vs irreversible inhibition of enzymes, must mention inhibition or inhibitor

  • reversible inhibitor of the enzyme

  • irreversible inhibitor of the enzyme

• biological reactions: metabolically reversible vs metabolically irreversible

Allosteric Enzymes Graph

• displays cooperatively: sigmoidal curve (Hill Eq)

• catalysis of one subunit increases/decreases catalysis of other subunits

• the inhibitors shift curve to the right: negative cooperation

• the activators shift cruve to the left: positive cooperation

Hill Kinetics Equation Breakdown

• θ: fraction of occupied sites where the ligand can bind to the binding site of the receptor protein

• [L]: free (unbound) ligand concentration

• K_a: apparent dissociation constant derived from the law of mass action (equilibrium constant for dissociation)

• Ka = Km: ligand concentration producing half occupation (ligand concentration, occupying half of binding sites), also microscopic dissociation constant

• n: Hill coefficient/number, describing cooperatively

Hill Kinetics: n

• n > 1: positively cooperative binding; once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules increases

• n < 1: negatively cooperative binding; once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules decreases

• n = 1: non-cooperative binding; the affinity of the enzyme for a ligand molecule is not dependent on whether or not other ligand molecules are already bound

Regulation of Activity of Allosteric Enzymes: Activation and Inhibition

• activators shift enzyme to the left

• inhibitors shift enzyme to the right