Enzymes: Biochemistry Introduction

Enzymes: Introduction

• Life depends on enzymes' ability to efficiently and selectively catalyze chemical reactions.
• Biomolecules are generally very stable, but reactions are too slow without enzymes to permit life.
• Enzymes enable acceleration, regulation, and coordination of chemical reactions.
• Catalytic power and specificity are the most notable features of enzymes.
• Enzymes prevent unhelpful or dangerous side reactions.
• Enzymes function as both information sensors and catalysts.

Enzymes: Vitalism

• Biochemical reactions were originally thought to be inseparable from life.
• Vitalism posits a fundamental difference and governance by non-physical elements for living vs non-living things.
• Prominent supporters of vitalism include Louis Pasteur.
• Eduard Buchner's experiment converting sugars into alcohol with dead yeast proved life's reactions were separate from life.
• Yeast's catalytic factor led to the term "enzyme," derived from the Greek for "in yeast".
• Eduard Buchner was awarded the Nobel Prize for this work 10 years prior to his death in WWI.

Enzymes: Co-Enzymes and Co-Factors

• Proteins are suited to form complex 3D structures, enabling a variety of substrates to bind.
• For certain enzymes, the protein component alone achieves activity.
• Other enzymes require co-factors, including inorganic ions, or co-enzymes, comprised of complex organic molecules, like vitamins.
• Prosthetic groups are co-enzymes or co-factors that are tightly associated with the enzyme.
• Different enzymes utilizing the same co-enzyme typically initiate similar reaction types.
• Apoenzyme + Co-factor/Co-enzyme = Holoenzyme

Catalysts: General

• Catalysts quicken reactions by lowering required amount of energy for reactions to proceed.
• Catalysts accelerate equilibrium attainment without changing the equilibrium itself.
• During reactions, catalysts are unchanged or recycled to participate in another reaction.
• Enzymes provide the rate enhancement of catalytic power.

Catalysts: Enzymes vs Chemical Catalysts

• Enzymes vs chemical catalysts
• Enzymes often achieve catalytic perfection, being faster than chemical catalysts
• Enzymes may function physiologically, whereas chemical catalyst require the extremes of temperatures, pressures, and pH levels.
• Enzymes include specificity, including stereospecificity.
• Enzymes are responsive to the dynamic needs of cells and organisms, which is unlike chemical catalysts.

Enzymes: Circe Effect

• Enzyme catalysis rates may approach the physical limit of the rates of diffusion of molecules in solution.
• Some enzymes have rate-determining steps that correspond to how fast substrates bind to the enzymes
• Several enzymes have reaction rates that surpass what diffusion-control limits predict.
• This phenomenon is named the Circe effect in reference to the enchantress in Greek mythology who drew her enemies to her and transformed them into animals.

Enzymes: Equilibrium and ES Complex

• Enzymes catalyze the interconversion of substrate and product.
• E + S ⇄ ES ⇄ E + P (enzyme-substrate complex)
• Substrate (S) corresponds to the molecule acted upon by an enzyme.
• Product (P) corresponds to the molecule produced by an enzyme.
• Active site describes the portion of enzyme (E) responsible for binding the substrate to form an enzyme-substrate (ES) complex.
• Enzymes converts substrate into product and can convert product into substrate. Since they can go both ways, they do not influence equilibrium, just the rate of equilibrium itself.

Enzymes: The Active Site

• The active site is a 3D cleft in the polypeptide chain.
• The active site is a small subsection of the enzyme.
• Active sites have their own unique microenvironments.
• Substrates attach to enzymes from several weak interactions.
• A precisely defined configuration of atoms drives specificity of substrate binding.
• During binding, enzymes and active sites can be flexible, causing both the "induced fit" or "conformation selection"

Enzyme Specificity: Lock-and-Key vs Hand-in-Glove

• The enzyme has to provide the desired environment to make the process or reaction likely to proceed.
• Within the lock-and-key model, specificity occurs btwn the 2 parts. This model doesn't explain for catalytic power, just specificity
• Induced Fit (Hand in Glove) model
• Demonstrates stereospecificity
• Conformational changes are occurring on both ends.

Enzymes: Free Energy (Rates and Equilibrium)

• For a reaction to be spontaneous, the change in Gibbs Free Energy (ΔG) must be negative, and releases energy (exergonic).
• Reactions with a positive ΔG will not occur naturally; input of free energy needed to drive these endergonic processes.
• A system at equilibrium exhibits zero net change in concentrations of reactants and products; ΔG is zero.
• ΔG of any reaction depends only on free energies of the product and reactant. Thus, ΔG is independent of transformation steps.
• ΔG gives no information about a reaction's rate of reaction; a reaction with a negative ΔG will take place spontaneously, yet has no influence on the rate at which takes place.

Enzymes: Free Energy (Rates and Equilibrium) Cont.

• Activation energy(ΔG‡) describes activation energy between substrates (S) and products (P). Determines the rate at which equilibrium is reached.
• Enzymes offer a lower-energy pathway between the product and substrate, while lowering the activation energy (ΔG‡).
• The relationship between reaction rate and activation energy is inverse and exponential.
• The free energy difference between the substrate (S) and the product (P) gives the reaction's equilibrium.
• Enzymes cannot influence the free energy difference between S and P

Enzymes: Rate Enhancements and Equilibrium

• Enzymes enable a lower-energy pathway btwn substrate and product, which lessens activation energy that enhances rate.
• Enzymes have no influence on free energies btwn substrate and product, which has influence on the equilibrium of the reaction
• Therefore, enzymes slow reactions down = Make slower rxns occur faster.

Enzymes: Modes of Enzyme Catalysis

• Catalytic capabilities of enzymes result from two reaction mechanisms
• Substrate Binding
  • Enzymes display both specificity in that they promote catalytic rate of reactions
  • Transition- state stabilization
• Chemical Effects
  • Acid/base catalysis encourages equilibrium
  • Covalent catalysis to lower E act of reaction rate
• To enhance equilibrium for increased rection rate, enzymes are lower E act pathway.

Binding Effects: Reaction Specificity and Catalysis

• The specificity and catalyzing power of subtrates provides specificity &catalytic power.
• Catalytic mechanisms limited to specific properties can still increase reaction rates by > 10,000x.
• E+S ⇄ ES ⇄ ETS ⇄ E + P
• Substrate Binding
• Transition-state Stabilization: Is where transition state is likely to occur.
• There is conceptual overlap between substrate binding and transition state stabilization.

Binding Effects: Substrate Binding

• Substrate binding is a "MATCH MAKER that promoting reactions in a REACTIVE way.
• Reducing entropy (decreased freedom of motion of two molecules in solution).
• Alignment of reactive functional groups of the enzyme with the substrate.
• Desolvation of a substrate(removal of water molecules) to expose reactive groups
• Distortion of substrates.
• Induced fit of an enzyme in response to substrate binding.

Binding Effects: Transition-State (TS) Stabilization

• There occurs increased interaction between an enzyme and substrate in the transition state.
• Stabilization of the transition state = essence of catalysis.
• An active site on each enzyme is complementary in shape and chemical traits.
• An enzyme distorts a substrate, guiding its transition state
• Enzymes possess the capacity bind at 1010 to 1015 times higher than other substrates
• Key traits an active site needs :must be similar enough to substrate to ensure specificity and different enough to promote change.

Transition-State Analogs: Competitive Inhibitors

• Transition-state analogs (TSAs) resemble unstable transition states.
• TSAs have potential therapeutic applications as competitive inhibitors = Stable 'mimick' of transition state".
• Competitive Inhibitors correspond to those molecules that can bind to the enzyme active site.
• It can stop substrate binding because it possesses a significant amount of affinity.

Enzymatic Catalysis: Chemical Effects

• After substrate binds enzyme can act upon substrate enhancing product formation
• The active site include reactive side chains
• Polar is important, plus ionizable Asp, Glu, His, Cys, Tyr, Lys, Arg, and Ser.
• Two are commonly used for processes of chemical catalyzation like acid/base catalytic and covalent.

Chemical Modes of Enzymatic Catalysis: Acid-Base Catalysis

• Catalytic transfer of a proton = reaction acceleration.
• The side chains of amino acids can either function as base (proton acceptors) or acid(proton donators).
• pH physiological level with Histidine can act as either acid/base catalysis
• functional group is influenced by the surrounding environment=Microenvironment.
• Amino acids possess a diverse set of pKa in function to what's around microenivornment.

Chemical Modes of Enzymatic Catalysis: Covalent Catalysis

• Subtrate is bound to enzyme to to help with reaction and create a reactive enzyme.
• A-X + E = X-E+A Stage 1 covalent linkage
• X-E + B = B-X + E Stage 2 get enzyme back to original form
• A-X = B = B-X+A overall reaction
• Example : a Group is transfered to both.
• Catalyzation is 2 steps the initial helps form a covalent with enzyme, 2nd regenrates freem enyzme.

Covalent Catalysis: Sucrose Phosphorylase

• Sucrose* + Pi <=> Fructose + Glucose-1-P
• *(Sucrose = disaccharide of glucose and fructose).
• Step one: transfer a glucosyl to an enzyme : Glucose- Fructose + Enz -> Glucosyl- Enz + Fuctose.
• Step Two: Glucose is transferred to enzyme: Glucosyl- Enz + Pi -> Glucosyl 1 phosphate + Glucosyl Enz.

Enzyme Kinetics: General

• Kinetics = study of velocity/rate of reactions. A velocity relates to concentration.
• The rate relates to alteration of products as time passes. V = Δ[P]/Δt. Always measured concentration in units of time

Enzymes Kinetics: Variables that Enzyme Velocity

• All enzymes are protiens and depend on the protein structure:
  • Activity of enzymes in temp is sensitive
  • At certain temperatures, enzymes have to unfold
  • certain optimum temperatures increase pH.

Enzymes Kinetics: Variables that Enzyme Velocity-Cont.

• Enzyme velocity is determined by enzyme and substrate
• With kinetics, there relies the relation of how these are dependent

Kinetics: Initial Velocity (Vo)

• Velocity explains the shift in concentration to get to equilibrium.
• Initial velocity equals the reaction at enzymes and can't measure Kinetics @ equilibrium.
• E+S -> ES - > E+P. Where V = k[] V velocity of transition

Michaelis-Menton Kinetics: Steady State Assumption

• Rate expression with the Michaelis and Menton
  • formation = the rate of its breakdown E+S= ES=EP [Es][k-1]+ [Es][k2]

Michaelis-Menten: Equation and Plot

• Substrate relations. km + [] =v

Michaelis-Menten: Km

• Rate relations + Km
• 1/2 of total substrate

Michaelis-Menten: Km Cont.

• When is too slow with not many substrate
  • Sensitive in concentration: When possesses high activity - With significant activity : - Km- Good range : is both high and good activity.

Michaelis-Menten: Sample Questions 1

• What is the velocity of reaction is = to Km ? v= at 2 mx

Michaelis-Menten: Sample Questions 2

• 2km =

Michaelis-Menten: Sample Questions 3

• Calculate km with 10x:1 km

Kinetics: Lineweaver-Burk Plots

• Linewever Burke describes [] and v of sub state. also measure of []

Kinetics: Enzyme Turnover Number

• Enzyme turner equal
  • Rate of /s

Reversible Enzyme Inhibition - General

• Controlling the enzyme : inhibitor
• Is prevented by Es and P

Reversible Enzyme Inhibition : competitive

enzyme binds = to substrate inhibitor

Reversible Enzyme Inhibition:-Uncompetitive-

Reversible Enzyme Inhibition Cont

Non competative with E+ + Is decrease

Chymotrypsin Cont. : serine

• Serine- breakdwon

Serine Protease :

• Has restricts and uniquely
• Enzyme activity

Serine Protease-Catalytic Triad

• Activity from Ser

Chymotrypsin mechanism Cont

• Acid base catalytic.

Enzyme Activity :

• Controls activities of activity .

Points Of Enzyme Activits :

• Reversibility with activities

Enzme activity points cont

• Activity with and to do with one another.

General Protiens

• Have slow movements to.
• Have diff structures in functions