Biochem Exam 2 (pt 2)

Enzymes produced by stomach of reminant mammals

Some early canteens were made from animal stomachs; when milk was stored in these canteens, it clumped into cheese

Chymosin is a protease enzyme that curdles the casein in milk

It is possible to convert glucose to ethanol via some action dependent on yeast

• Substances that increases the rate of a reaction 
• Not consumed in the process
• Do not affect thermodynamic equilibrium (they don't make the products more favorable, only accelerate the transition

• Most enzymes are proteins or protein-RNA complexes 
• Ribozymes are RNA catalysts

• Higher reaction rates- typically 10⁶ to 10¹² times faster than uncatalyzed reactions
• Milder reaction conditions: below 100 degrees C. normal atmospheric pressure, and often near neutral pH
• Greater reaction specificity

• Concentration of substrates and products 
• or
• Regulating enzyme activity via: allosteric control, covalent modification, variation of enzyme amount

The part of the enzyme which binds the substrate, and contains the residues that directly participate in making and breaking bonds

A reactant, acted upon by the enzyme

Substance that reduces or enhances the activity of an enzyme

Small molecule required for enzyme activity (not for all enzymes)

Active enzyme bound to required cofactors

The enzyme without required cofactors

One reaction cycle

• Catalyzes conversion of CO₂ and H₂O to carbonic acid
• One of the fastest rxns

Factor by which reaction rates are enhanced by an enzyme= catalyzed rate/uncatalyzed rate

• Enzymes vary in their ability to distinguish between substrates
• Some are very specific and only accelerates with one substrate
• Most enzymes can act on a range of similar substrates i.e alcohol dehydrogenase (oxidizes small alcohols)

• Oxidoreductases
• Transferases
• Hydrolases
• Lyases
• Isomerases
• Ligases

Redox reactions

Transfer of functional groups

Hydrolysis reactions

Group elimination to form double bonds

• isomerization reactions
• converts from one isomer to another

Bond formation coupled with ATP hydrolysis

Transferases for a phosphate group

Hydrolases for phosphoryl groups

Hydrolases for amide bonds in proteins

If a reaction has a negative change in Gibb's then you know a reaction can occur (favorable) but only kinetics tells you how quickly

Rate of reaction is defined by this which is required to transit a via a highly unstable transition state

• The transition state is energetically unfavorable (high free energy) complex
• The larger the change in energy of the transition state, the slower the reaction rate
• -Transition state is unstable, therefore it is very small in the reaction mixture

Transition state will quickly decompose to reactants or to products

• The slowest step: typically the formation of transition state
• In a multi step reaction, the step with the largest activation energy is the rate limiting step

Reactions can be accelerated by temperature (T)

• The lower the change of Gibbs at the transition state, more molecules have enough energy to cross the activation barrier
• Enzymes provide a reaction pathway with a lower activation energy in order to reach equilibrium more quickly
• 

Bell-shaped curve

They stabilize an intermediate state- compound state similar to the transition state

• Substrate entropy reduction: proximity, orientation
• Preferential binding of the transition state
• Acid-base catalysis
• Covalent catalysis
• Metal ion catalysis

• Enzyme-catalyzed rxns use standard organic mechanisms that are more efficiently than in solution
• Proximity (local concentration) and orientation (proper alignment) are two obvious physical properties that can be manipulated by an enzyme

Bringing reactants close together and with the enzyme catalytic center and aligning them for reaction speeds up reaction

• Rate enhancement: up to 10⁷-10⁸
• Large entropic penalty that must be compensated by binding energy of the enzyme-substrate complex

• Enzyme-substrate complex
• Driven by free energy/affinity (inherent)
• Energy barrier: solvation/desolvation and changes in structure/association (induced fit)

• Reflected in energy barrier between S and ES states
•  
• First step is the formation of the enzyme-substrate complex (ES complex)
• ES complex is typically more favored than free E and S due to enthalpic forces
• EP complex is typically slightly less favored than free E and P to potentiate release of the product

Energy difference for (Change G_cattransition state) for catalyzed reaction (ES^transition-ES) is less than Change in G noncatalyzed transition state (S^transition-S) for a reaction that is enhanced by transition state stabilization

• Enzymes are not optimized for strong bonding to their substrates
• Enzymes should (bind well) to transition states
• 

• Multiple weak interactions between enzyme and substrate are responsible for ES complex
• These weak interactions cover the "cost" of forming energetically unfavorable transition state

Substrate is biased towards transition state in the bound enzyme-substrate complex (apply steric strain)

Typically a covalent complex between enzyme and substrate

Increases the rate of the catalyzed reaction

2 addtl H bonds between enzyme and transition state relative to substrate results in ~10⁶ rate enhancement

Reactions can be catalyzed by proton transfer or sharing between a substrate and an enzyme (acid-base chemistry)

Requires a side chain that can donate or accept a proton

Ensures the enzyme is returned to its original state at the end of the reaction allowing the enzyme to process another substrate

• Typical dome-like shape
• Optimal pH for most enzymes match their environment

Enzymes may accelerate reaction rates through transient formation of enzyme-substrate covalent bonds

Often involves reaction of a nucleophilic group with an electrophilic substrate (i.e sometimes called nucleophylic catalysis)

• Reaction coordinates of covalent catalysis
• The more stable the intermediate, the harder it is to break the covalent bond

The rate limiting step requires more energy to break

Metalloenzymes require a metal ion for catalytic activity

• Fe²⁺
• Fe³⁺
• Cu²⁺
• Mn²⁺
• Co²⁺
• Mo²⁺

• Na⁺
• K⁺
• Ca²⁺

• Zn²⁺
• Mg²⁺

• Binding to substrates to orient them for reactions
• Oxidation-reduction reactions by changing the metal ion oxidation state
• Electrostatic stabilization or shielding of negative charges

Metal ions can make covalently bound water molecules more acidic than free water: good source of catalytic OH-

• Proximity, orientation, and entropy reduction
• Preferential binding of the transition state complex (transition state stabilization)
• Acid-base catalysis
• Covalent catalysis

• Chymotrypsin
• Trypsin
• Elastase
• etc.

• The amide bond is very stable and difficult to break:
• Standard temperature/environment: half-life ~20 years
• Uncatalyzed: boil overnight in 6M HCl
• Catalyzed: protease activity at physiological pH and temp continually breaks down proteins

covalent chemical bond that can be broken by an enzyme

aromatic hydrophobic residues

Basic residues: Lys, Arg

• Ala, Gly, and Val
• but primarily Ala

• Catalytic domain of the Nuclear Inclusion-A protein from tobacco etch virus (TEV)
• 

• Coagulation cascade factors that enable blood clotting
• 

• Intestinal enzyme that cleaves inactive forms (zymogenes) of other proteolytic enzymes (e.g. chymotryspinogene, proelastase)
• 

• Filled by the tetrahedral intermediate, but cannot be accessed by the planar carbonyl of the amide bond
• Amide protons from Gly193 and Ser195 make 2 additional hydrogen bonds to the tetrahedral transition state leading to significant stabilization of the of the transition state

Nonspecific proteases that are tightly controlled by their production in an inactive form that requires activation

Enzymes produced in their inactive forms

Chymotrypsinogen, trypsinogen, and other zymogens are synthesized in the pancreas and secreted into small intestine

Intestinal enteropeptidase cleaves trypsinogen to start an activation cascade

• Similar cascades are involved in blood coagulation
• Deficiency in one of these coagulation factor proteases that disrupts the activation cascade causes hemophilia (poorly clotting blood leading to profound bleeding)

• Competitive serine protease inhibitor
• Forms stable complexes with and blocks the active sites of enzyme
• Binding is reversible

• Trypsin, chymotrypsin, but also plasmin and plasminogen activator involved in thrombolysis
• Used in surgeries as a drug (aprotinin) that inhibits thrombolysis

Side effects: blood clotting (thrombosis)

• Study of reaction rates
• Simplest of reactions: S(reactant) -> P (product)

• Reaction rate is equal to the disappearance of S and the formation of P over time (t)
• 

• E+S -> ES complex -> EP complex -> E+P
• With high [S], reaction is zero order with respect to S
• 

• Formation of product is the rate limiting step, therefore ES complex is formed promptly and EP ignored
• 

• If we only consider initial velocity (v₀), at early time points P=0 and K_-2=0
• 

• moles/second product formation at reaction initiation
• substrate is consumed as reaction proceeds, so [S] changes with time leading to an observed change in velocity
• 

If [S]>>[E]t, so the fraction of S that binds to E (to form ES) is negligible, and [S] is constant at early points

• Since k₂ is the rate-limiting step (k_-1>>k₂), the formation and breakdown of ES quickly reaches equilibrium and [ES] is constant
• v₀=k₂[ES]

When [S] is high, at saturation: [ES]~[E]tot, and maximum reaction velocity 

v_max=k₂[E]_Tot

• 
• Equilibrium constant that has units of concentration
• K_m is the dissociation constant for the ES complex
• Km is the substrate concentration ([S]) at which v₀=v_max/2

A large K_m represents weak binding and a smaller K_m represents stronger binding

• The steady-state assumption is in effect change in [ES]/change in t=0
• k₂ is the rate-limiting step and v₀=k₂[ES]
• At saturation: [ES]~[E]_Tot, and maximum reaction velocity v_max=k₂[E]_tot

• Under these conditions:
• 

Also referred to as Double-Reciprocal Plots

• Use the Michaelis-Menten equation to form the line
• 

• Catalytic constant: the turnover number or the number of reaction processes catalyzed per unit of time
• k_cat=v_max/[E]_Tot

Rxn is faster when k_cat is higher

K₂

• k_cat/K_m
• A good measure of catalytic activity because it takes into account the rate of catalysis (k_cat) and the enzyme-substrate interaction (K_m)

Means the compound is a better substrate for the enzyme

Represents the ability of an enzyme to convert substrate into product

• a non-michaelis-menten reaction
• ACD covalently crosslinks actin into non-functional species
• Also binds to one molecule of ATP and two molecules of actin

• Allosteric regulation (metabolism, signaling, etc.)
• Drugs (e.g. antibiotics, anti-cancer drugs)

Can be reversible and irreversible

• Competitive
• Uncompetitive
• Noncompetitive
• Mixed

• The inhibitor competes with the substrate for binding to the enzyme active site
• Transition state analogs are very strong competitive inhibitors
• 

• Binds only after the substrate is bound
• Works best when substrate concentration is high

• Binds to an allosteric site, does not affect affinity for a substrate
• 

• Binds to an allosteric site, affects affinity for a substrate
• 

Irreversibly modify a catalytically important active site residue

Can be nonspecific and specific

Inhibitors can be targeted towards reactive groups with specific activity: DIPF (diisopropyl phospho-fluoridate, DFP, DIFP) reacts specifically with serine residues

In serine proteases, only one Ser is located in the active site and is sufficiently activated to react, other serine residues will not react

• Irreversible covalent modification can be combined with substrate recognition to generate a specific irreversible inhibitor
• Also known as reactive substrate analogs

• Reversible inhibitors bind to an enzyme, but can be removed
• Each class effects enzyme kinetics differently

Will not change since high substrate concentrations will eventually out-compete the inhibitor for binding

• Inhibition is affected by the KI (the KD for the enzyme-inhibitor complex) and [I]
• -Smaller KI= tighter binding= more inhibition
• 

• Where the product of the enzyme binds to the active site and down-regulates enzyme activity
• A common form of competitive inhibition (negative feedback)

The factor by which [S] must be increased to overcome the inhibitor

Can lead to blindness and death but is treated with large amounts of ethanol

The ethanol out-competes methanol for binding to alcohol dehydrogenase, preventing the conversion of methanol to toxic formaldehyde

Uncompetitive inhibition, the inhibitor binds directly (and exclusively) to the ES complex

Inhibitor affects the catalytic function of the enzyme, but does not perturb substrate binding to the enzyme (S binding is improved)

• 
• 

• Mixed and noncompetitive inhibition
• The equilibrium distinguishes noncompetitive and mixed inhibition

Special case where K_m with inhibitor=K_m without inhibitor is noncompetitive inhibition