BIOS 452 Exam 3

lactose

galactose + glucose

maltose

glucose + glucose

sucrose

glucose + fructose

D-fructose

ketone, C3

glucose

aldehyde, C3

D-mannose

aldehyde, C2, C3

D-galactose

aldehyde, C4, C3

ribose

aldehyde, 5 carbon, none on left

enzymes

• catalyze biochemical reactions without being used up

• typically globular

• structure is essential to catalytic activity

native conformation

primary, secondary, tertiary, and quarternary structure very important for catalytic activity

enzymes and functional groups

• enzymes position functional groups so reaction occurs more easily

enzymes and energy

• lower the activation energy (energy of transition state), but not overall difference in energy of products and reactants

cofactors

inorganic ions, coenzymes

inorganic ions

Fe²⁺, Mg²⁺, Mn²⁺, Zn²⁺

coenzyme

complex organic or metalloorganic molecule (coenzyme) — like vitamin

apoenzyme

the protein part of enzyme that has cofactor

holoenzyme

apoenzyme + cofactor

• catalytically active enzyme

enzymes and the body

• diseases caused by no/deficient/excessive enzyme activity

• measures of enzyme activity can be used to diagnose illness

• drugs act through interactions with enzymes

• part of practical tools in cheme, food tech, agriculture

enzymes in biochemistry

• make things

• change things (phosphorylate)

• signalling

• metabolism

• enzyme inhibitors

biocatalysts over inorganic catalysts

• more specific (avoid side products)

  • enzymes will always make the most favorable product where an inorganic catalyst may also make minor products

• milder reaction conditions

• control biological pathways

• metabolites can decompose in a variety of ways, enzymes make sure most desirable way is the one that proceeds

oxioreductase

transfer of electrons (hydride ions or H atoms)

NAD+ → NADH = reduction

NADH → NAD+ = oxidation

transferases

transfer groups

• adding phosphate group

hydrolases

hydrolysis reactions — transfer of functional groups to water

• breaking peptide bond

lyases

cleaves of bonds (C-C, C-O, C-N, etc.) by elimination (leaving double bonds, rings) or addition (adding groups to double bonds)

• pyruvate → acetaldehyde

isomerase

transfer of groups within molecules to yield isomeric forms

malate → fumarate

ligases

formation of (C-C, C-S, C-O, C-N) bonds by condensation reactions coupled to cleave of ATP or similar cofactor

pyruvate → oxaloacetate

how do enzymes work

create a specific environment which speeds up reaction

• substrate interacts with specific group of Amino acids (may included cofactor and coenzyme) in active site

substrate

molecule bound in active site and acted on by the enzyme

active site

• pocket where enzyme-catalyzed reaction occurs

• often, active site encloses substrate (isolating it from solution)

• surface is lined with AA that help bind substrate and catalyze reaction

delta G^o

standard free energy change (at pH 7) from S→P

high activation energy

slower reaction

sucrose breakdown

sucrose breakdown is favorable but high activation energy — why are our bodies so good at it? enzymes!

reaction intermediates

transient chemical species

rate-limiting step

• step in enzymatic reaction with highest activation energy

• determines rate of reaction

energy barrier bw S and P

energy is needed to

• align reacting groups (align functional groups)

• formation of transient unstable charges

• bond rearrangements

• other transformations needed for reaction to proceed in either direction

how is activation energy lowered

• weak, noncovalent interactions in ES complex

  • h-bonding

  • hydrophobic interactions

  • ionic interactions

• sometimes stabilize transition state (not just in ES complex)

delta G_B

binding energy: delta G uncatalyzed - delta G catalyzed

• when ES forms, small amount of free energy is released and stabilizes complex

  • each favorable non-covalent interaction releases a little energy

• used by enzymes to lower activation energy of reactions

• lower substrate entropy or cause conformational change in enzyme (induced fit)

how enzymes lower activation energy

• active sites bind transition states better than original substrate

• stronger/additional interactions with transition state

• energy to change ES → transition state is partly compensated for by the fact that transition state is the favorable conformation in the active site (this lowers the energy of the transition state between ES and EP)

  • binding energy is the energy released from transition state being the most favorable conformation

transition state analogs

many drugs are transition state analogs — same structure as transition state so they occupy enzyme to stop some reaction from happening

enzyme specificity

enzyme can differentiate between its substrate and similar molecule

(eg it can differentiate D vs L enantiomers)

• specific enzyme has one or few substrates

substrate selectivity

substrate can be acted upon by one or more enzymes

induced fit

• when substrate binds, weak interactions lead to conformational change in enzyme (fit to transition state not substrate)

• achieves:

  • brings specific functional groups into proper position to help with reaction

  • produces weak interactions in transition state (stabilizing)

  • new enzyme conformation has enhanced catalytic properties

binding energy contributes to reaction specificity and catalysis

1. entropy reduction

   1. binding energy holds substrates in proper orientation to react

2. desolvation of substrate

   1. enzyme-substrate interactions replace substrate+water interactions — raises environment entry since water is “less ordered”

3. binding energy compensates thermodynamically

   1. for electron redistribution the substrate undergoes in order to react

   2. essentially, binding energy subsidizes the reaction

4. binding energy used to cause conformational change in enzyme itself when substrate binds (induced fit)

   1. alignment of specific functional groups

   2. formation of additional weak interactions in transition state

additional catalytic mechanisms (involve covalent interaction)

• acid-base catalysis

• covalent catalysis

• metal ion catalysis

once S bound to E

• ES complex undergoes catalytic mechanisms that may involve transient covalent interactions with substrate or group transfer to or from substrate

general acid-base catalysis

give and take protons

• if no catalyst present, charged intermediates break down immediately and products never get formed

• so, these catalysts stabilize intermediates via proton transfer — these intermediates break down into products

covalent catalysis

• transient covalent bond forms between enzyme and substrate

change reaction paths (new pathway has lower activation energy) than uncatalyzed reaction

• uncatalyzed: A-B → w/H₂O → A + B

• catalyzed: A-B + X → A-X + B → w/H₂O → A + X + B

requires nucleophile on enzyme

metal ion catalysis

use redox factors, pKa shifters

• ionic interactions between enzyme-bound metal and substrate

  • orient substrate for reaction

  • stabilize transition states

1/3 of enzymes use metal ions

specific acid-base catalysis

uses only H+ and OH- from water

general acid-base catalysis

proton transfers by other classes of molecules like AA side chains

non-enzymatic reactions

if in aqueous solution (not enzyme), weak organic acids or bases can be proton donors in addition to water

acid-base catalysis in active site

• amino acid side-chains are proton donors and acceptors

(general acid-base catalysis bc water not present for specific acid-base catalysis)

proton transfers

most common biochemical reactions

covalent catalysis nucleophiles

reactive

• serine (S)

• thiolate (C)

• amine (N, Q)

• carboxylate (D, E)

units for each concentration

[E] ~ nM

[S] ~ microM to mM

[S] is 5-6 orders of magnitude as much as [E]

• during first 60 seconds of reaction, [S] can be considered constant

@ low [S]

• V₀ increases almost linearly

• [S] < [E] so V₀ rises as [S] rises

@ high [S]

• V₀ plateaus as enzyme is oversaturated with substrate

• [S] > [E] so rising [S] makes no difference. [E] is fully saturated so V₀ plateaus at V_max

Michaelis-Menten Kinetic Scheme

E + S ←→ (fast) ES ←→ (slow) E+P

K_m

Michaelis constant

steady state assumption

rate of ES formation = rate of ES breakdown

lineweaver burk — x axis

1/[S] mM^-1

lineweaver burk — y-axis

1/V₀ = 1/ (microM/min)

lineweaver burk — x intercept

-1/K_m

lineweaver burk — y intercept

1/V_max 1 / (microM/min)

V_max

• lineweaver burk plot is better indicator because V_max is plotted, vs in michaelis menten curve, it’s estimated

K_m

• different between enzymes

• different for different substrates for the same enzyme

• (k₂ + k_-1) / k₁

k₂ <<< k_-1

K_m = k_-1 / k₁ = K_d

So, K_m is measure of affinity between enzyme and substrate

(higher K_m = lower affinity)

k_cat

• rate constant for rate limiting step

• turnover number — number of substrate molecules that are converted to product over a given time on one enzyme molecule when enzyme is saturated with substrate

  • (max substrate → product one enzyme can do in a given time when substrate is readily available)

specificity constant

• k_cat / K_m

• measure of catalytic efficiency

• overall conversion rate of E+S → E+P

• unit = s^-1M^-1

multisubstrate

ternary, pingpong

ordered ternary

E + S₁ ←→ ES₁ ←→ ES₁S₂ → E + P₁ + P₂

(in random ternary, it’s like a diamond — either ES₁ or ES₂ first, then meet at E₁S₁S₂ → E + P₁ + P₂

ping-pong double displacement

E + S₁ ←→ ES₁ ←→ E’P₁ ←→ E’ ←→ E’ + S₂ ←→ E’S₂ → E’ + P₂

aspirin

inhibits enzyme that catalyses first step in synthesis of prostaglandins (involved in some processes that produce pain)