Biochemistry Lecture Notes: Focus on HIV-1 Protease Mechanisms and Aspartic Proteases - Lectures 18 and 19

what are the mechanisms for catalysis to remember?

- catalysis by proximity

- covalent catalysis

- general acid-base catalysis

- metal ion catalysis

- low barrier hydrogen bonds

what occurs in catalysis by proximity?

- enzymes that are close enough with proper orientation have collisions with higher frequencies

- active site is pre-organized to form near-attack complexes

what occurs in near-attack complexes?

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

- they have 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?

motion

what can active site conformations 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 occurs in covalent catalysis?

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

- facilitates electron transfer

- most mechanisms are unknown

where does covalent catalysis usually occur?

- nucleophilic attack by amino acid side chains

- involves prosthetic groups (cofactors)

what is general acid-base catalysis?

transfer of a proton in the transition state

what can transferring a proton do?

- activate nucleophiles

- stabilize charged groups

- improve electrostatic interactions that stabilize transition state

what does specific signify?

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?

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

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 like zinc, magnesium , or iron lose electrons thus existing as cations (ions with positive charge)

what can cations do?

- stabilize transient and intermediate structures

- assist in forming strong nucleophilic species

- hold substrate inside the active site

- stabilize charge

what occurs in low-barrier H bond?

- typical strength is about 10-30 kJ/mol

- O-O separation of about 0.28 nm

- as distance becomes smaller, H bonds become stronger

- pKa values must be similar in two electronegative atoms

- assist in catalysis

- stabilization energies can approach 60 kJ/mol

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?

digestive enzymes secreted as proenzymes/zymogens

- all cleave polypeptide chains

- require cleavage to be active

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

- the specificities are different (different active sites)

what is thrombin?

blood clotting enzyme

what is subtilisin?

bacterial enzyme

what is a tissue plasminogen activator?

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

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

- chymotrypsin is aromatic, so its pocket has hydrophobic serine

- 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

what are burst kinetics?

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

what occurs in the serine protease mechanism?

there's a mixture of 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

how is a tetrahedral oxyanion intermediate stabilized?

NH groups of glycine and serine

what does general base catalysis by histidine form?

E-Ser-S covalent intermediate

(refer to slides 14-15 on lecture 19 to understand mechanism)

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 oxygen ion in the tetrahedral oxyanion is stabilized by interaction with the backbone 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, Cos, or Thr)

what are aspartic proteases?

- active site contains two aspartic acids

- cleave peptide bond BETWEEN two hydrophobic amino acids

- NO COVALENT CATALYSIS

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

HIV-1 protease is a homodimer

pepsin is a monomer

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

a catalytic aspartate to the active site

what does the mechanism of aspartic proteases require?

one Asp that is protonated and another that is deprotonated

what is peak performance?

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?

1. catalytic water

2. tetrahedral intermediate

3. Who's fitting the bill- LBHBs

what do LBHBs allow for in aspartic proteases?

hydrogen tunneling

what do HIV-1 proteases cleave?

polyprotein products of the HIV genome

- 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?

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