lecture 5: cysteine proteases

how does cysteine (thiol) proteases degrade proteins

uses a common catalytic mechanism based on a nucleophilic cysteine thiol

what are cysteine proteases used for generally?

• protein degradation

• apoptosis

• survival of parasites

• maturation of viruses

what was the first cysteine protease that had its structure discovered?

papain, comprised of 2 domains and a cleft with a catalytic dyad (cys/his pair)

why is cysteine a stronger acid than serine

serine is more electronegative than cysteine. cysteine has a lower pKa than serine so it can be more easily deprotonated

cysteine protease mechanism

why is the hydrolysis of a peptide bond similar to the deamination of an amide

they both use a cys/his dyad to promote the hydrolysis of a C-N bond

Burkholderia pseudomallel causes what and how

• causes whitmore’s disease (found in contaminated soils/ water)

• BPSL1549- the toxin (deamidase) has no sequence similarity to any outside protein on Burkholderia

how is BPSL1549 similar to cytotoxic necrotising factor 1 (CNF1)

it shares a similar structure (but not sequence) (conservation of the his/cys pair)

what is CNF1

a toxin expressed by some pathogenic strains of E.coli that deamidates a key glutamine in the family of small GTPases. this blocks GAP-dependent deactivation of these GTPases by inhibiting GTP hydrolysis, leading to the remodelling of the actin cytoskeleton

what is mandelate

the substrate of mandelate racemase, a member of the enolase superfamily that catalyses the inversion of the chiral centre of mandelate using acid-base catalysis

what does enolase superfamily enzymes use to facilitate catalysis

an enzyme-bound magnesium ion

explain the structure of the enolase superfamily

• residues involved in acid/base catalysis are found in the barrel domain

• substrate binding residues are found at the interface of the capping and barrel domains → 2 subgroups:

1. MR- mandelate racemase group

2. MLE- muconate lactonising enzyme group

what is an enolate anion

a negatively charged intermediate formed by deprotonation of the alpha-carbon adjacent to a carbonyl group, stabilised by resonance between the carbon and oxygen atoms

metal ion catalysis and the mechanism of madelate racemase

the active site of mandelate racemase

the magnesium ion that stabilises the acid-carboxylate is coordinated by multiple carboxylic acid groups of nearby asp/glu residues.

the L-proton faces the sidechain of lys166 with the catalytic histidine that can return to the D-proton on the opposite side