Exam 2 Content - Biochem

enzymes

a class of proteins that has the extreme ability to enhance reaction rates 10^6-7 fold; are highly specific to substrates; do NOT change the overall favorability of the reaction, just change how quickly the reaction can occur

stabilize the transition state by interacting more with the TS than with the substrate (lowers free energy of TS)

delta G under standard conditions

represents the change in free energy under standard conditions assuming equilibrium; describes the intrinsic favorability of a reaction, neglecting actual cellular conditions

delta G under cellular conditions

equal to the delta G under standard conditions plus the product of R (gas constant), temperature, and the natural log of product concentration over reactant concentration; shows the favorability of a reaction given the current cellular conditions, not assuming equilibrium

binding energy

the free energy released upon an enzyme substrate complex to an enzyme-TS complex; is always favorable when an enzyme stabilizes the TS

active site

a region on an enzyme where the substrate/TS interacts; the R-groups in this site are typically far away in the enzyme’s primary structure and it makes up a small part of the entire enzyme; typically capable of excluding water (many nonpolar R-groups, also can include polar/charged R-groups); stabilizes through many weak interactions; high complementarity to the substrate

Michaelis Menten kinetics

a model to describe simple enzymatic reactions (though most reactions are bisubstrate, it is often assumed that only one is rate limiting); only provides an estimate of Vmax and Km

shows positive log relationship between reaction velocity and substrate concentration; demonstrates that at low [S] the relationship is approximately first order and that at high [S] the relationship is approximately zero-order (saturation)

Km

in Michaelis Menten kinetics is the substrate concentration at which the reaction velocity is half of the maximum reaction velocity; lower values are more impressive

constructing MM graph

1) Prepare multiple reactions with constant [E] and varying [S], and monitor/plot the rate of product formation against time for each [S]

2) Determine the initial reaction velocities (slope of [P] vs. time graphs) for each [S]

3) Plot each [S] against its own Vo

Lineweaver Burke plot

also a double reciprocal plot, provides a linear relationship for Vo and [S]

is the reciprocal of Vo against the reciprocal of [S], where slope is Km/Vmax and y-intercept is 1/Vmax

kcat

describes how quickly the product is formed per unit time; the reaction velocity for one copy of enzyme (Vmax normalized by [E]); the “turnover rate” for an enzyme

catalytic efficiency

kcat/Km; due to the values not being inherently correlated, a well scoring enzyme will have a high kcat and low Km

Vo in first-order region

cell typically keep [S] at or below Km such that Vo is equal to catalytic efficiency times [E] times [S]

sequential binding

when all substrates bind an enzyme before the product is released, forming a ternary complex; can be random where either S binds first or ordered where a specific S must bind first

double displacement

where one product is released from an enzyme before the second S binds; the enzyme is transiently covalently modified

enzyme inhibitors

molecules that can act as poison or useful tools to bind covalently or noncovalently to an enzyme and modify its effectiveness; can be reversible to illustrate Km and Vmax

competitive inhibitors

molecules that directly compete for the S-binding site on an enzyme; can not form an ESI-complex

the relative [I] and [S] will determine what binds to E; does not alter Vmax but increases Km

no change to y-int on LB plot, increases slope (no change because Km increases while Vmax doesn’t change)

uncompetitive inhibitors

when S binds E and creates a new binding site for I to bind; can NOT have I binding without ES-complex; forms ternary ESI-complex

decreases Km because ES-complex consumed to form ESI-complex, so ES complex will more readily form (Le Chatelier’s); also decreases Vmax by creating fewer functional copies of the enzyme (essentially lowering [E])

increased y-int on LB plot because Vmax decreases, no change to slope because Km and Vmax decrease proportionally

noncompetitive inhibitor

when a molecule binds an enzyme no where near the S-binding site and causes a deformation of the S-binding site, but does not inherently affect S-binding

no change in Km because I binds independently of S (can form EI or ESI-complex), reduces Vmax because there are less functional copies of E

increased slope on LB plot because Vmax decreases, increased slope because Km doesn’t change while Vmax decreases

covalent catalysis

when the active site has a reactive group (that is typically a nucleophile) that can temporarily covalently bind to a part of the substrate (seen in chymotrypsin mechanism)

general acid-base catalysis

when a molecule (other than water) acts as a H+ donor/acceptor (seen in chymotrypsin mechanism)

metal ion catalysis

when a metal ion serves as a bridge between the enzyme and substrate, holding the substrate in the appropriate conformation to optimize binding energy (seen in DNA polymerase)

catalysis by approximation

when two substrates are brought together on a single binding surface to enhance reaction rate (seen on ribosomes)

protease

a class of enzyme that catalyze peptide bond hydrolysis

peptide bond hydrolysis

a thermodynamically favorable but kinetically slow (without enzyme) reaction that involves breaking down polypeptides into free amino acids; extremely slow due to stabilization of peptide bonds from partial double bond behavior

chymotrypsin

a type of protease enzyme that specializes in cleaving peptide bonds that follow the carbonyl of amino acids with large, hydrophobic side chains

chymotrypsin catalytic triad

three amino acid residues in chymotrypsin that work together to catalyze peptide bond hydrolysis just after large, hydrophobic R-groups; includes Ser195 (nucleophilic attacker), His (acid-base catalyst), and Asp (maintains orientation of His)

chymotrypsin mechanism

1) E binds S → pocket region of enzyme filled with substrate R-group, aligning C of C=O to be attacked by Ser195; more favorable contacts between R-group and enzyme increase binding energy

2) E attacks S → Ser195 is deprotonated by His to form alkoxide ion that attacks C=O of peptide, forming and acyl-enzyme complex with tetrahedral intermediate (sp2 C=O becomes tetrahedral)

3) oxyanion hole of enzyme stabilizes the tetrahedral intermediate in TS

4) TI collapses into first product (R—NH2) that leaves (His donates proton for amine group), and acyl-enzyme at Ser195 (covalently modified)

5) His deprotonates a water molecule that acts as a nucleophilic attacker for acyl-enzyme at Ser195

6) oxyanion hole stabilizes new tetrahedral intermediate and His donates proton again, but to Ser195 to regenerate enzyme

7) second product (with carboxyl) leaves and enzyme has completed one turnover