MBIO 2700 / Topic 6a: Enzymes

> What does a catalyst do?

> What are the usual compounds in biochem = catalysts?

> Speeds up rate of rxn w/o being consumed.

> Usual compounds = catalysts = enzymes.

> What are enzymes?

> Are enzymes limited to proteins?

> Enzymes = Broad class of molecules catalyzing biochemical rxns.

> Proteins ≠ always the enzymes.

> What is an apoenzyme? What does it consist of?

> What is an holoenzyme? What does it consist of?

> Apoenzyme = Inactive form of enzyme = protein w/o cofactor.

> Holoenzyme = Active, complete enzyme = apoenzyme + cofactor.

Differentiate the 6 classes of enzymes based on their ability.

> Oxidoreductases = Carries in/away e^- into/from a molecule (often in form of H⁺).

> Transferases = Carries groups between molecules.

> Hydrolases = Adds H₂O to cleave.

> Lyases = Forms/adds dbl bonds to cleave.

> Isomerases = Isomerize by group transfer.

> Ligases = Forms C-C, C-S, C-O, C-N bonds (using energy from ATP hydrolysis).

> What is responsible for the majority of all biochemical reactions? How so?

> Do enzymes produce side reactions? Why/why not?

> Is the activity of enzymes uncontrollable?

> Enzymatic activity = Responsible for biochemical rxns → Enzymes regulate these reactions.

> Enzymes are specific to their path of rxns that they want to happen → No side rxns.

> Enzymes can be continually produced but turned off when not needed and back on when needed.

Differentiate the following terms: substrate (S), enzyme-substrate (ES) complex, active site (AS).

• S = Specific reactant molecule upon which E converts into products (Ps).

• ES = Non-covalent, reversible association formed when S binds to AS of E.

• AS = Specific region of E where S binds.

> What is the generic uncatalyzed reaction equation?

> What is the generic enzyme-catalyzed reaction equation?

> S → S‡ → P

> E + S → ES → ES‡ → EP → E + P

> What does the transition state (TS) theory say?

> What’s a TS?

> Rxn rates ≈ energy needed to get reactant to TS.

> TS = highest-in-energy configuration of a molecule on a rxn pathway.

> What is the activation energy (∆G‡/E_a)?

> Why is S‡ higher than S?

> What does S look like in ES? Why does it look like this?

> E_a = energy needed to get S to S‡.

> S must go through distortions, such as bond-stretching, -breaking, -locking in rare conformation, et cetera, to react.

> S is starting to look like S‡ in ES‡ → S in ES is partly distorted.

What are the 3 things that determines whether or not E and S will bind?

E recognizes + binds to S if:

> S fits into AS complementary to TS.

> S correctly matches specific non-covalent interactive sites in AS.

> Either (a) S can conform to AS or (b) E can conform to S.

Can E generally catalyze more than one reaction using a ton of substrates?

E catalyzes one rxn using a min # of S.

How do enzymes exhibit specificity with respect to optical and geometric isomers?

> Optical specificity = D-isomer of S can be accepted by E but its L-isomer can’t.

> Geometric specificity = trans isomer of S can be accepted by E but its cis isomer can’t.

What are the four things that happen when the ES complex is formed?

> S into AS = Order increases = S decreases = G increases.

> S hydration shell cut to bind to AS.

> E adjusts to shape of S‡.

> S groups and E groups are aligned to react.

What happens when the enzyme-substrate complex is overly stabilized due to perfect substrate binding in the ground state?

> S fits perfectly into E + all non-covalent interactions have occurred in ES.

> Ground state ES becomes too stable + (TS peak w/ this enzyme = TS peak w/o enzyme).

> Now requires more energy to destabilize it to reach TS.

> Very difficult to reach TS.

> S not distorted.

> Few to no products.

What happens when the enzyme-substrate complex is efficiently stabilized due to imperfect substrate binding in the ground state?

> S doesn’t fit perfectly into E + not all non-covalent interactions have occurred in ES.

> Ground state ES not too stabilized + (TS peak lowered w/ this enzyme ≠ TS peak w/o enzyme).

> Requires less energy to destabilize it to reach TS.

> Relatively easier to reach TS.

> S can be distorted.

> More products made.

What is the relationship between binding energy and TS?

> To get to ES‡ = Energy required.

> Energy released from non-covalent interactions = Binding energy.

> E complementary to TS (not S) → Not all non-covalent interactions made in ground-state ES but @ TS → These interactions stabilize TS.

> Energy required to reach TS = Energy released when all non-covalent interactions occur = Binding energy.

The interactions (a) lower the energy of TS → lower E_a → speeds up reactions and (b) release energy to get to TS.

What are the three things that binding energy is also used for when climbing to ES‡ dagger?

> Overcoming reduction of entropy = Ordering the S into AS.

> Overcoming solvation effects = Cutting bonds btwn hydration shell around S.

> Overcoming strain = Reducing any steric and/or electronic strain.

Does K_eq get affected by catalysts? Why or why not?

> Catalyst → speeds up fwd and rev rxns equally → lowers E_a for both.

> (fwd rate : rev rate) = Determines position of equilibrium → unchanged.

> Catalyst doesn’t affect position of eq or K_eq.

How does the chymotrypsin mech work? Do explain the condition of the enzyme before and after.

> E compresses for S to fit → compress Asp¹⁰² and His⁵⁷ H-bond → enhanced interaction changes His from an acid to a base by raising pK_a

We get our first T_d intermediate via:

> His⁵⁷ transfers H from Ser¹⁹⁵ R group.

> (-) charged O of Ser¹⁹⁵ R group attacks CO of S.

> pi bond electrons of CO of the substrate gets transferred to O of CO.

We get our first T_d collapse via:

> T_d collapsing via O pi bond restoration and kicking off the N terminus of S.

> (-) charged N terminus transfers proton from His⁵⁷.

> Electrons that once held N and H together in His⁵⁷ returns to N.

We get our second T_d intermediate via:

> His⁵⁷ transfers proton from H₂O.

> (-) charged O of deprotonated H₂O (now ^-OH) attacks the CO of S.

> pi bond electrons of CO of S gets transferred to the O of CO.

We get our second T_d collapse via:

> T_d collapsing via O pi bond restoration and kicking off the Ser¹⁹⁵’s -OR.

> (-) charged O of leaving -OR transfers proton from His⁵⁷.

> Electrons that once held N and H together in His⁵⁷ returns to N.

> In the end, we get two products: one being the molecule cleaved after the aromatic residue and second being the substrate that contains the aromatic residue.

> Free enzyme is now the state we have, not compressed. So His⁵⁷ and Asp¹⁰² is not compressed, and His⁵⁷ is now acidic again.

What is stabilized in the oxyanion hole and what stabilizes what it stabilized? Hint: There are two different ways it stabilizes what it stabilizes at different points of the mech.

> O is stabilized with the alpha-amines of Gly¹⁹³ and Ser¹⁹⁵ only when O is negatively charged, a.k.a. during T_d int.

> O is stabilized only by amine of Ser¹⁹⁵ when in its CO form.

What does the hydrophobic pocket contain, and what does the pocket do?

Contains the aromatic part of the R group to hold S during mechanism.

Why on earth study enzymatic mechanisms to such a degree? Explain the way you’d like to.

> Knowledge of how biochemical reactions happen en vivo.

> Knowledge of how to modify or regulate said reactions.

> Artificial synthesis of biologically relevant compounds to said mechanism.

What are the three key ways that metallic cofactors help in enzymatic activity?

> Interactions between metals and S help stabilize charged transitions states, intermediates, and substrates:

+ If ES‡ has (-) portions within it → stabilized by metal cations; if ES‡ has (+) portions within it → stabilized by metal anions.

> Interactions between metals and S help orient and binding of S to E.

> During enzymatic activity, the metals can act as e^- shuttles = Carrying e^-’s from one part of the activity to another part of the activity → Help in streamlining overall activity.