Kinetics

Chapter 8
Biomedical Importance
• Enzyme kinetics is the quantitative measurement of the rates of
enzyme-catalyzed reactions and factors affecting these rates.
• Required for homeostasis
• Describe each step of a reaction and details of its mechanism.
• Clinical indicators in blood tests.
• Enzyme therapy
Balanced Equations
• Lists the substrates and stochiometry
• Products, P and Q, refer to reactants that are thermodynamically
favored.
• Reversible
• Irreversible
• Reactions can also occur rapidly rendering the products unable to
participate in the reverse reaction, or functionally irreversible under
physiologic conditions.
Changes in Free Energy Determine the
Direction
• Gibbs free-energy change describes the direction of a chemical
reaction.

• If the free energy of products is lower than substrates, G is negative,

and the reaction is spontaneous.
• The sign and magnitude of each part of the formula is important.
• G provides information about the direction and equilibrium, not the
rate.
Rates are Determined by Activation Energy
• Transition states are important in understating thermodynamics.

• It is the simultaneous bonding of group L to the substrate and product.

• Changes in G are associated with partial reactions.

• The overall G represents the number of transition states.

• Overall thermodynamics does not tell anything about mechanism or
kinetics.
GF Defines the Activation Energy
• Majority of GF of chemical reactions are positive meaning there are
energy barriers called activation energy.
• The frequency of overcoming this barrier is inversely related to Eact
such that GF determines how fast is the formation of transition states.
Factors Affecting Reaction Rate
• Kinetic Theory/Collision Theory states that:
• Molecules must approach within bond-forming
distance
• Must possess sufficient kinetic energy to
overcome the barrier
Factors Affecting Reaction Rate
• Temperature
• Increased kinetic energy
Factors Affecting Reaction Rate
• Reactant Concentration
• Increased chance to collide with double rates if A or B is doubled.

• The kinetic order of the reaction is equivalent to the sum of the

stochiometric coefficients of the substrates.
Factors Affecting Reaction Rate
• Pseudo-first order conditions refer to solutions wherein one substrate
is significantly in excess over the other making it virtually constant.
• In these conditions, the rate of reaction will depend solely on the
variable reactant or the limiting reactant.
Keq is a ratio of Rate Constants
• All reactions are reversible to some degree and at equilibrium and
additionally, reactants and products have the same concentration.
Keq is a ratio of Rate Constants
• Properties of systems at equilibrium
• Equilibrium constant is a ratio of the reaction rate constants, not reaction
rates.
• At equilibrium, the reaction rates of forward and reverse reactions are equal.
• The value of Keq can be calculated from the concentrations of substates and
products OR from the ratio k 1/k-1.
• Equilibrium is dynamic and can be detected using radiolabeled substrates.
Enzymes Lower the Activation Energy Barrier
• Enzymes accelerate rates by lowering GF for the formation of
transition states as well as stabilizing these states.
• Stabilization can be achieved by the following:
• Suitable position of acid-base groups for proton transfer
• Suitable position of charged groups that stabilize the charges
• Causing steric strain to approach transition state.
• Some enzymes have a unique mechanism due to covalent catalysis
with more favorable Eact.
Enzymes do not Affect Kq
• Enzymes are unchanged after the reaction it catalyzed.
• They do not affect the G0 of the overall reaction.
Factors of Enzyme-Catalyzed Reactions
• Temperature
• Increased Temp increases kinetic energy.
• However, excess Temp can denature the enzyme which loses that
catalytic activity.
• Most enzymes are stable up to 45-55oC.
• Temperature coefficient (Q10) is the rate of increase for a 10 oC
increase in Temp.
• In homeotherms, changes in reaction rates due to Temp occurs only
in special circumstances such as fever or hypothermia.
Factors of Enzyme-Catalyzed Reactions
• H+ concentration. pH affects charge of the enzyme and substrate.
• Significant contribution to reactions.
• Most enzymes are stable between pH of 5-9.
Assays Typically Measure the Initial Velocity
• Initial rate conditions approximate a short time period of enzyme-
catalyzed reactions during measurement.
• Negligible amount of products is present.
• Initial velocity vi is the rate of forward reaction.
• Assays use pseudo-first order condition with respect to the enzyme.
Substrate Concentration Affects Rate
• Under pseudo-first order conditions, the behavior of a multisubstrate
enzyme will imitate an enzyme with a single substrate.
• Substrate concentration increase vi until a maximum value Vmax
wherein the enzyme is saturated.
Michaelis-Menten Equation
• The Michaelis constant Km is the substrate concentration at which vi is
half the Vmax at a given concentration of enzyme.

• At S much less than Km:

Michaelis-Menten Equation
• At S much greater than Km:

• At S equal to Km
Linear form of Michaelis-Menten Equation is
used to Determine Km and Vmax
• Achieving the high concentrations of S to reach Vmax is impractical.
• Extrapolation from vi using a linear form if MME circumvents this
impracticality.
• The final equation is similar to the equation for straight line:

y = ax + b
Linear form of Michaelis-Menten Equation is
used to Determine Km and Vmax
• Lineweaver-Burk plot can be used to determine the kinetics of an
enzyme inhibitor.
• However, avoid using low 1/S values to avoid bias, ie. in solutions with
excessively large amounts of substrates, vi becomes Vmax.
Catalytic Constant
• Some parameters may be used to compare the activity of enzymes or
different preparations.
• Impure enzyme preparations are typically expressed in specific
activity (Vmax/protein concentration)
• For homogenous enzymes, the activity of the enzyme is expressed as
the catalytic constant kcat if the number of the active sites is known.
Catalytic Efficiency
• High kcat is only beneficial if Km is sufficiently low.
• Recall that an enzyme with LOW Km has a HIGHER affinity for the
substrate.
Catalytic Efficiency
• Some enzymes have instantaneous reactions or substrates are rapidly
converted to products.
• The rate-limiting step in these enzymes is the formation of the ES
complex.
• These enzymes are called diffusion-limited or catalytically perfect.
• Examples include triosephosphate isomerase, carbonic anhydrase,
acetylcholinesterase and adenosine deaminase.
Catalytic Efficiency
• In cells, enzymes can be arranged in complexes to streamline each
step until the final product is made and released to the solution.
• Example is fatty acid synthetase.
Km may Approximate a Binding Constant
• Affinity of an enzyme to a substrate is inverse of the dissociation of
the ES complex
Hill Equation Describes Enzymes that exhibit
Cooperative Binding
• An exclusive property of multimeric enzymes wherein a bound
substrate increases the affinity of binding to additional substrates.

• MME cannot be used to describe cooperative kinetics.

• When S is low relative to k’, the initial reaction velocity increases.
• The slope of the line, n, is the Hill coefficient.
• Depends on the number, kind and strength of interactions of binding sites.
• When n = 1, all sites are independent and MME is observed.
• When n > 1, the enzyme has positive cooperativity.
• Higher n, higher degree of cooperativity
Kinetic Analysis Distinguishes Competitive
from Noncompetitive Inhibition
• The interaction between an inhibitor and enzyme depends on forces
important in protein structure and ligand binding such as H bonding
and hydrophobic interactions.
• Inhibitors can be classified based on the following:
• site of action on the enzyme
• whether they chemically modify the enzyme
• Kinetic parameters them influence.
• Transition state analogs
• Mechanism-based analogs
Competitive Inhibitors Resemble Substrates
• Can be overcome by increasing the substrate.
• The inhibitor binds to the substrate-binding portion of the enzyme
• A competitive inhibitor acts by decreasing the free enzyme molecules
available for the substrate.
• Increasing S raises the reaction and can overcome the inhibitors and
also depends on the following:
• Inhibitor concentration
• Affinity for the enzyme
• Km
• An example is succinate dehydrogenase inhibited by malonate.
Double-Reciprocal Plot Facilitates Evaluation
of Inhibitors
• Used to distinguish competitive and non-competitive inhibitors.
• vi is measured at different substrate concentrations with and without
inhibitors.

• Ki, or the constant with inhibitor, can be calculated when Km is known.

• Ki is used to determine different inhibitors of the same enzyme.
• Lower Ki means a more effective inhibitor.
Double-Reciprocal Plot Facilitates Evaluation
of Inhibitors
• When 1/S approaches 0, vi is independent from the inhibitor.
• Thus, a competitive inhibitor has no effect on Vmax but raises the
apparent Km (K’m).
Noncompetitive Inhibitors Lower Vmax but
does not affect Km
• Does not affect binding of the substrate
but rather, decreases Vmax with the
same Km.
• These inhibitors bind at sites different
from the substrate binding sites.
• There is no structural similarity with the
substrate.
 Dixon Plot
• Alternative to the Lineweaver-Burk plot.
• vi is measured at different inhibitor concentrations
with constant substrate.
• This is used to compare potency of inhibitors in
pharmacology.
IC50
• Concentration of inhibitor with 50% inhibition usually measured by
mixing with an ES solution until 50% of the substrate is displaced.
• Varies depending on the environment of the solution.
Tightly Bound Inhibitors
• Some inhibitors have high affinity such that the inhibitor
concentration required to Ki falls below the concentration of enzyme
in an assay.
• More inhibitors are bound compared to that of assumed in a steady
state.
• These require specialized equations to estimate Ki or IC50.
Irreversible Inhibitors
• There is chemical or permanent modification of the enzyme.
• Covalent bonds are made or broken with amino acids generally
involved in substrate binding, catalysis or maintenance of functional
conformation.
• Enzymes are modified and removing the inhibitor does not
reconstitute the enzymatic activity.
Mechanism-Based Inhibition
• Also known as Suicide Inhibitors
• Contains a group that is catalyzed by the enzyme.
• Forms a covalent bond and blocks the function of an essential
residue.
• Enzyme specific and unreactive outside the enzyme’s active site.
• Note that these enzymes are not characterized by Lineweaver-Burk or
Dixon plots since the activity of the enzyme does not decrease.
Most Enzyme-Catalyzed reactions have more
than one Substrate
• Fundamental principles are similar to enzymes with single substrates
but the mathematics to evaluate a multimeric reaction is complex.
Sequential or Single-Displacement Reactions
• Sequential reactions require both
substrates to bind to the enzyme to
form a ternary complex before
catalysis.
Most Bi-Bi conform to MME
• Vmax is the reaction rate when both substrate are present at
saturating concenctrations.
• Each substrate has its own Km – S concentration of SA that yields half
Vmax if the SB is at saturating levels.
• Double reciprocal plots can still be used to determine Vmax and Km.
Ping-pong mechanism Double Sequential
Enzyme Kinetics and Drug Development
• Pharmacology aims to identify:
• Drugs the eradicate or prevent the progression of pathogens - antibiotics
• Stimulate endogenous defense mechanisms - vaccines
• Halt molecular processes triggered by genetic, environmental or biologic
stimuli - steroids
Enzyme Kinetics for Drug Screening
• To select assay condition for the presence of inhibitor.
• Concentrations of substrate should be adjusted properly to detect the
enzyme’s activity without masking the presence of an inhibitor.
• Kinetics provides means for quantifying and comparing potency of
and mechanisms of inhibitors.
• Noncompetitive inhibitors are desirable since they cannot be
overwhelmed by substrate concentrations.
Most Drugs are Metabolized in vivo
• Drugs need to resist degradation by enzyme present in the patient or
pathogen, or drug metabolism.
• With penicillins, some pathogens have the enzyme β -lactamase
which metabolize the drug. Concomitant administration of β-
lactamase inhibitor prevent that metabolism.
• Prodrugs are inactive form of drugs that require metabolism to take
effect.
Thank You