PPT Enzyme Kinetics - Dr. Bonleon Flashcards
The quanititative measurement of the rates of enzyme-catalyzed reactions.
Enzyme kinetics
*Systematic study of factors that affect these rate.
Permit the reconstruction of the number and order of the individual steps which enzymes transform SUBSTRATE to PRODUCTS.
Enzyme Kinetics
Identify potential drugs that selectively enhance or inhibit the rates of specific enzyme-catalyzed processes.
Enzyme kinetics
*Reveal details of the catalytic mechanism
Lists the initial chemical species (SUBSTRATES) present and the new chemical species (PRODUCTS) formed for a particular reaction.
Balanced chemical equation
All in their correct proportions.
Stoichiometry
By kinetic and chemical modification, the central questions about enzymes:
- How do they work?
2. How do they catalyze reactions
A typical overall enzyme-catalyzed reaction involving a single substrate and a single product.
Enzyme kinetics
Reaction occurs in both directions
Double arrows
___________ provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction.
Enzymes
_____________ chemically facilitates CATALYSIS.
Active site
Direction in which a chemical reaction will tend to proceed.
Gibbs free energy
Concentrations of reactant and products that will be present in equilibrium.
Gibbs free energy
🔼Gp - 🔼Gs =
🔼G or Gibbs free energy
Free energy that accompanies transition from the standard state, one-molar concentrations of S & P, to equilibrium.
🔼G0
🔼G0 : 🔼G0 at a standard state of 10-7 M protons at pH ______.
7
If the free energy of the product is LOWER than that of the substrates.
Negative
Direction LEFT TO RIGHT.
Negative
*Spontaneous
________ & ________ of free energy change determine how FAR the reaction will proceed.
Sign and magnitude
Independent of the mechanism of reaction.
🔼G0
🔼G0 provide information only about the ___________ & ___________ state of the reaction.
Direction and equilibrium
Provides no information concerning the rates of reaction.
🔼G0
We can use the sign of 🔼G0 to figure out whether a reaction is spontaneous in the forward direction, backward direction or if the reaction is at ______________.
Equilibrium
Virtually all chemical reactions have an energy barrier separating the reactants and the products.
Barrier : free energy of activation
Energy difference between that of the reactant and a high-energy intermediate that occurs during the formation of the product.
Free energy of activation
Sufficient energy needed for molecules to react.
Rate of reaction
Energy to overcome the energy barrier of the transition state.
Rate of reaction
Only a small portion of the molecules may possess enough energy to achieve the transition state between reactants and products.
No enzymes
Determined by the number of energized molecules.
Rate of reaction
The _______ the free energy of activation, the more molecules have sufficient energy to pass through the TRANSITION STATE = the FASTER the rate of reaction.
Lower
A reaction may be looked at as passing from one valley, representing __________, over a mountain pass to another valley, the ___________.
Stable reactants
Products
The pass between them is called the ______________, the state from which the molecules may with equal probability go ahead to products or back to reactants.
Transition state
Energy must be put into a reaction to raise the reactants to the top of the pass, the transition state; this energy is called the _______________.
Free energy of activation or activation energy
The role of the catalyst is to find A LOWER PASS over the mountain range, a pathway with a _____________.
Lower activation energy
An alternative way of looking at the role of a catalyst is that is ___________ the transition state.
Stabilizes
Provides the enzyme allowing a reaction to proceed rapidly under conditions prevailing in the cell.
Alternate reaction pathway
Condition of Alternate reaction pathway:
Lower free energy of activation
Enzyme does not change the free energies of the reactants or products and doe not change the equilibrium of the reaction.
Alternate reaction pathway
For 2 molecules to react they must approach within BOND-FORMING DISTANCE of one another (collide).
Kinetic theory or Collision theory
For two molecules to react they must possess SUFFICIENT KINETIC ENERGY to overcome the energy barrier for reaching the transition state.
Kinetic theory or Collision theory
Anything which INCREASES the frequency or energy of collision between the substrates will INCREASEZ the rate of reaction in which they participate.
Kinetic theory
Factors that affect the reaction rate:
Temperature and reactant concentration
Raising the ambient temperature __________ the kinetic energy of molecules.
increases
If the concentrations of BOTH A and B are DOUBLED, the probability of collision will ________.
Increase fourfold
If concentrations of EITHER A and B are doubled, the probability of collision will _________.
Double
At equilibrium, the overall concentrations of reactants and products remain constant.
Keq: ratio of rate constants
The rate of conversion of substrates to products therefore equals the rate at which products are converted to substrates.
Keq: ratio of rate constants
___________ is a ratio of the reaction rate constants.
Equilibrium constant
Reaction rates of the forward and back reactions are equal
Equilibrium
No net change in the concentration of S & P.
Equilibrium
*Dynamic state
Individual S & P molecules are continually being interconverted.
Equilibrium
The environment of the active site _______ 🔼Gf by stabilizing the transition state intermediates.
Lowers
______________ suitably positioned to transfer protons to and from the developing transition state intermediate.
Acid-base groups
*Suitably positioned charged groups or metal ions that stabilize developing charges.
Imposition of ___________ on S that their geometry approaches that of the transition state.
Steric strain
Factors that affect the rate of enzyme-catalyzed reactions:
Temperature and hydrogen ion concentration
________ temperature will INCREASE rate of both catalyzed and non-catalyed reactions by increasing kinetic energy & collision frequency of the reacitng molecules.
Increase
_________ can also increase the kinetic energy of the enyzme to the point that exceeds the energy barrier for disrupting their noncovalent interactions that maintain the enzyme’s 3D structure __________.
Heat
Denaturation
Loss of catalytic activity.
Denaturation
Enzymes from humans generally exhibit stability at temperatures up to _______ to ________.
45 - 55C
Clinical importance: FEVER or HYPOTHERMIA
Intracellular enzymes exhibit optimal activity at pH:
5-9
Reflects balance between denaturation at high or low pH.
Hydrogen ion concentration
Residues involved must be in the appropriate state of protonation for the reaction to proceed.
Acid-base catalysis
Most common charged groups:
- Negative carboxylate groups
2. Protonated amines
Gain or loss of critical charged groups adversely affect substrate binding and thus will __________ or _________ catalysis.
Retard or abolish
Catalytic activity may require that an amino group of the enzyme may be in the __________ form.
Protonated form
At __________ ph, the group is DEPROTONATED thus DECREASING the rate of reaction.
Alkaline
Extremes of pH
Denaturation
Structure of catalytically active protein molecule depends on the _____________ of the amino acid side chains.
ionic character
Effect of enzyme concentration on reaction velocity
Michaelis-Menten kinetic theory
Michaelis-Menten kinetic theory: If the substrate concentration is held CONSTANT, velocity of the reaction is __________ to the enzyme concentration.
Proportional
Effect of substrate concentration on reaction velocity:
[S] is _______, v is 1st order with respect to substrate.
Low
Effect of substrate concentration on reaction velocity:
[S] is HIGH, reaction is ____________ (independent of S)
Zero order
Effect of substrate concentration on reaction velocity:
Mid-[S], reaction is __________ (proportionality is changing).
Mixed-order
At LOW VALUES of S, the initial velocity Vi rises almost ________ with increasing S.
Linearly
But as S INCREASES, the gains in Vi level off forming a ____________.
Rectangular hyperbola
The substrate concentration that produces a Vi that is one-half of Vmax is designated the ____________.
Michaelis-Menten constant
________ is roughly an inverse measure of the affinity or strength of binding between the enzyme and its substrate.
Km
The LOWER the Km, the _______ the affinity (so the lower the concentration of substrate needed to achieve a given rate).
Greater
Used because it is difficult to estimate Vmax from the position of the asymptote (plot of a rectangular hyperbola).
Lineweaver-Burk linear plot
Plotting the reciprocals of the SAME DATA POINTS yields a “double reciprocal” or _____________. This provides a more precise way to determine Vmax and Km.
Lineweaver-Burk plot
Is determined by the point where the line crosses the 1/V1 = 0 axis so the [S] is infinite.
Vmax
Km = Vmax times the slope of line. This is easily determined from the intercept of ________.
X-axis
[S] is very _______ compared with [E] where when all E are bound, there is still an excess of S.
Large
Rate of breakdown of ES = rate of formation of ES
Steady state assumption
Characteristic of an enzyme and its substrate.
Km
Reflects the affinity of the enzyme for that substrate.
Km
Numerically equal to the substrate concentration at which the reaction velocity =
1/2 vmax
Does not vary with the concentration of the enzyme.
Km
High affinity of the enzyme for the substrate.
Small Km
Lower concentration of the substrate is needed to half-saturate the enzyme.
Small Km
Low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme.
Large Km
Rate of reaction is DIRECTLY PROPORTIONAL to the enzyme concentration.
Velocity-enzyme concentration
If enzyme concentration is HALVED, the initial rate of reaction and Vmax is reduced to half that of the original.
Velocity-enzyme concentration
Inhibitors compete directly with substrate for binding to the active site (i.e catalytic site).
Competitive inhibition
Overcome by raising the concentration of the substrate.
Competitive inhibition
Inhibitors bind only to the ES complex at a site distinct from the active site (i.e the allosteric site).
Uncompetitive inhibition
Inhibitors bind both to the free enzyme and to the ES complex at the allosteric site, which is distinct from the active site.
Noncompetitive inhibition
Bind covalently so tightly to the active site that the enzyme is inactivated irreversibly.
Irreversible competitive inhibitors
Does not obey the Michaelis-Menten kinetics.
Irreversible competitive inhibitors
Substrate analogs that possess a highly reactive group that is not present on the natural substrate.
Affinity labels
The active site is PERMANENTLY BLOCKED from the substrate because the group reacts covalently with amino acid residue.
Affinity labels
The reside modified - not necessarily involved in catalysis.
Affinity labels
Substrate analogs that are transformed by the catalytic action of the enzyme.
Mechanism-based or suicide inhibitors
Product of this reaction is HIGHLY REACTIVE which combines covalently with amino acid residue in the active site resulting to ENZYME INACTIVATION.
Mechanism - based or suicide inhibitors
Substrate analogs whose structures closely resemble the transition state of the natural substrate.
Transition state analogs
Do not covalently modify the enzyme but bind the active site so tightly that they irreversibly inactivate it.
Transtition state analogs
Provide pharmacologic agents and research tolls for study of mechanism of enzyme action.
Enzyme inhibition
Highly toxic, naturally-occuring and man made compounds.
Irreversible enzyme inhibitors
