Biochem Exam 3

what are the mechanisms for catalysis to remember?

catalysis by proximity

covalent catalysis

general acid-base catalysis

metal ion catalysis

low barrier hydrogen bonds (LBHBs)

what occurs in catalysis by proximity? (enzymes)

enzymes that are close enough with proper orientation have collisions at higher frequencies (more often)

the active sites in catalysis by proximity are

pre-organized to form near-attack complexes

in catalysis by proximity what is the percentage of absence and presence of enzyme

Absence: 0.0001%

Presence: 1 to 70%

what do atoms in near-attack complexes do?

reacting atoms are in van der Waals rxns contact at an angle resembling the bond to be formed in the transition state

how are near-attack complexes characterized as

reacting atoms within 3.2 A and an approach angle of +/- 15 degrees of the bonding angle in the transition state

what's an example of a near-attack complex?

alcohol dehydrogenase

what's essential for enzyme catalysis?

protein motions

what kind of motions happen in enzyme catalysis

bonds vibrate, side chains bend and rotate, backbone loops wiggle and sway, and whole domains move as a unit

what can active site conformation changes do?

assist substrate binding

bring catalytic groups into positions (orientation)

induce formation of NACs

assist in bond making/breaking

facilitate conversion of substrate to product

what do in the active site residues do in covalent catalysis?

form a temporary covalent bond with the substrate. At the end of the rxn, the covalent bond is broken to regenerate the enzyme

where does covalent catalysis usually occur and what does it involve?

nucleophilic attack by amino acid side chains

involves prosthetic groups (cofactors)

what happens with electrons in covalent catalysis

facilitates electron transfer

most mechanisms are unknown

what is double displacement

when two substrates bind and react separately in a ping pong manner

what is E’

covalently modified enzyme intermediate

what is general acid-base catalysis do?

transfer of a proton in the transition state

what can transferring a proton (H+) do?

activate nucleophiles

stabilize charged groups

improve electrostatic interactions that stabilize transition state

what does specific signify in general acid-base catalysis?

H+ or OH- that has diffused into the active site

what are the candidate amino acids for acid-base catalysis?

glutamic acid, aspartic acid, and histidine

how does histidine play a role in acid-base catalysis?

histidine can be deprotonated by another group and then act as a base, accepting a proton from the substrate

how does water play a role in acid-base catalysis?

can act as a acid or base at the active site through proton transfer with an assisting active site residue

what are the primary amino acids in enzyme active sites?

histidine, cysteine, aspartic acid, glutamic acid, arginine, lysine, tyrosine, serine, threonine, asparagine, glutamine

what do secondary roles do?

engage directly in catalytic effects in enzyme active sites

half of the amino acids

what are some examples of secondary roles?

raising/lowering pKa values

orientation of catalytic residues

charge stabilization

proton transfers via hydrogen tunneling

what occurs in metal ion catalysis?

metal atoms lose electrons thus existing as cations (ions with positive charge)

example metal atoms in metal ion catalysis

zinc, magnesium , or iron

what can cations do? (metal ion catalysis)

stabilize transient and intermediate structures

assist in forming strong nucleophilic species

hold substrate inside the active site

stabilize charge

what is the bond strength of H-bonds and O-O separation

H-bonds: 10-30 kJ/mol

O-O separation: 0.28 nm

LBHBs what happens as the distance between heteroatoms become smaller (<0.25nm)

H bonds become stronger

what does pKa need for LBHBs to happen

pKa values of the two electronegative atoms must be similar

What helps assist catalysis in terms of LBHBs

energy released in forming the LBHBs

in LBHBs H-bonds become

shorter and thus stronger

what are some different types of serine proteases?

trypsin, chymotrypsin, and elastase

thrombin

subtilisin

plasmin

tissue plasminogen activator

what are trypsin, chymotrypsin, and elastase and what are they secreted as?

digestive enzymes secreted as proenzymes/zymogens

what does trypsin, chymotrypsin, and elastase do

all cleave polypeptide chains

what are trypsin, chymotrypsin, and elastase similarities

they all have similar sequences, structure, and mechanisms which are all important for function

what are trypsin, chymotrypsin, and elastase differences

the specificities are different (different active sites)

what is thrombin?

blood clotting enzyme

what is subtilisin?

bacterial enzyme

what does tissue plasminogen activator do?

it cleaves plasmin proenzyme plasminogen and is administered to prevent heart attack

is acetylcholinesterase a protease?

no, but it is mechanistically similar in breaking down acetylcholine due to covalent catalysis

what is the catalytic triad?

histidine, aspartic acid, and serine. these three make up the powerhouse

where does trypsin cleave?

carbonyl side of arginine and lysine (basic AAs)

what AA can NOT be cleaved?

proline, it's very kinky

where does chymotrypsin cleave?

carbonyl side of phenylalanine and tyrosine (aromatics)

Where does elastase cleave?

carbonyl side of small, neutral residues like glycine, alanine, and serine

in substrate-binding pockets, what determines specificity?

nature of the pocket

what is trypsin nature of pocket

trypsin is basic, so its pocket has negatively charged aspartic acids

what is chymotrypsin nature of pocket

chymotrypsin is aromatic, so its pocket has hydrophobic serine

what is elastase nature of pocket

elastase is small, so its pocket has bulky residues like threonine and valine (branched)

how is chymotrypsin kinetics assayed?

artificial substrate

nitrophenolate product absorbs at 400 nm

burst kinetic

what are burst kinetics?

the first step is very fast, the second step is really slow

do serine proteases display burst kinetics

yes

there's a mixture of what in the serine protease mechanism?

covalent and general acid-base catalysis

in serine protease mechanisms, what does aspartic acid do?

it orients histidine by forming an LBHB

in serine protease mechanisms, what does histidine do?

acts as a general acid and base (like water)

in serine protease mechanisms, what do covalent bonds do?

turns a trigonal C into a tetrahedral C

in serine protease mechanism what is a tetrahedral oxyanion intermediate stabilized by?

backbone NH groups of glycine and serine

in serine protease mechanism what are the steps to chymotrypsin breaking down a protein

substrate binding the aromatic side chain fits into hydrophobic pocket (substrate arrives and settles into the pocket)

general base catalysis by histidine to form E-Ser-S covalent intermediate

Histidine is stabilized by a LBHB

Tetrahedral intermediate collapse and P (product) is released

Stable intermediate makes way for water

Nucleophilic attack by water-Histadine as a general base

Tetrahedral intermediate collapse and P is released

Active site ready to repeat

what does the chymotrypsin mechanism involve?

two tetrahedral oxyanion transition states

how are transition states stabilized in the chymotrypsin mechanism?

a pair of amide groups known as the oxyanion hole

what is the oxyanion hole?

the negative oxygen ion in the tetrahedral oxyanion is stabilized by interaction with the backbone (NH) amide groups of serine and glycine

where are catalytic triads found?

several hydrolyses and transferase enzymes

how do catalytic triads occur?

divergent and convergent evolution

what do catalytic triads include?

acid to orient and stabilize the base (Asp, Glu, His)

base to polarize the nucleophile (His or Lys)

nucleophile to attack the substrate (Ser, Cys, or Thr)

how are aspartic proteases different from the serine ones

different structure and mechanism

in aspartic proteases what are in the active sites

two aspartic acids (Pepsin residues 32 and 215)

what does aspartic proteases do

cleave peptide bond between two hydrophobic amino acids

do aspartic proteases have covalent catalysis

no

what are the structures of HIV-1 protease and pepsin respectively?

homodimer and monomer

what does each lobe contribute to for HIV-1 protease and pepsin?

a catalytic aspartate to the active site

what is aspartic proteases dependent on and active at

dependent on pH profile

active at acidic pH

what does the mechanism of aspartic proteases require?

one Asp that is protonated and another that is deprotonated

what is peak performance and what is it dependent on?

the optimal activity

dependent on acid and base (Asp's)

why is the observed pKa of pepsin much smaller than the normal pKa?

the microenvironment

what are the three important things to know about the mechanism of aspartic proteases?

catalytic water

tetrahedral intermediate

Who's fitting the bill- LBHBs

what do LBHBs allow for in aspartic proteases?

hydrogen tunneling

what does HIV-1 proteases do?

cleave polyprotein products of the HIV genome

What can HIV-1 proteases do in terms of mammalian aspartic proteases

remarkable imitation of mammalian aspartic proteases

because HIV-1 protease is a homodimer, this means it's

more genetically economical for the virus

what is the active site of the HIV-1 protease strucutre?

it's two-fold symmetric; different flaps

what's an example of a protease inhibitor?

AIDs drugs

If HIV-1 protease can be selectively inhibited, then

new HIV particles cannot form (no envelope protein)

what has structure based drug design done?

developed several inhibitors that work in the dish

what was the inhibitor Crixivan made by?

Merck

how are enzymatic reactions regulated?

to match cell requirements

how is enzyme regulation achieved?

abundance and activity

what is abundance controlled by?

gene expression (not treated)

what kind of phenomenon is enzyme regulation?

natural and physiological

what is the velocity of a reaction typically controlled by?

concentrations of substrates and cofactors

Review: What are cofactors?

metal ions or organic coenzymes that participate in some enzyme reactions

what reduces the velocity of the reaction?

accumulation of product (negative feedback)

when is there no further reaction apparent?

once the ratio of [P]/[S] approaches Keq, it causes an increased rate of the reverse reaction

what does it mean when an enzyme is allosterically regulated?

substrate and product bind elsewhere. This causes the activity to change

what are the different ways that enzyme regulation can occur?

covalent modification

zymogen (proenzyme) activation

isozymes

control by modulatory proteins

allosterically

what can covalent modification of side chains do?

it can activate or inactivate an enzyme

what are the possible AAs that can act in covalent modification?

serine, threonine, tyrosine, aspartate, and histidine

(most common ones are those with hydroxyl groups)

the enzymes that introduce and remove modifications can be

regulated (reversible)

what are the 2 ways enzymes can be regulated

allosteric control or covalent modification

how can the generation of products be controlled?

by controlling the ratios between active and inactive enzymes