Proteases

Question 1

How do enzymes increase the rate of chem rxns?

Answer 1

lowers the activation energy;
stabilizes/binds to the transition state more tightly than the ground state of the substrate
(Tooze 205, 207)

Question 2

typical suffix for enzymes

Answer 2

-ase

Question 3

alt name for proteases

Answer 3

proteinases, peptidases

Question 4

what are proteases?

Answer 4

any enzyme that performs proteolysis (hydrolysis of peptide bonds)

Question 5

4 functional families of proteases

Answer 5

serine, cysteine, aspartic, metallo

Question 6

how to classify proteases based on the 4 functional families of proteases?

Answer 6

identify the nature of the most prominent functional group in the active site

Question 7

mammalian chymotrypsin and bacterial subtilisin are what kind of proteases?

Answer 7

both are serine proteases

Question 8

what is a scissile bond?

Answer 8

a covalent bond that can be broken by an enzyme; eg peptide bond of a substrate cleaved by a protease

Question 9

what do serine proteases do?

Answer 9

cleaves peptide bonds to produce smaller peptides

Question 10

first step of serine protease rxn

Answer 10

acylation:
covalent bond produced btwn substrate’s C1 and enzyme’s serine’s -OH group

neg charged tetrahedral (about C1) intermediate becomes an acyl-enzyme intermediate

part of peptide is released

Question 11

second step of serine protease rxn

Answer 11

deacylation:
acyl-enzyme intermediate hydrolyzed by water, restores serine’s -OH group

neg charged tetrahedral (about C1) intermediate

second peptide released

Question 12

side chains in serine protase

Answer 12

serine 195, histidine 57, and aspartate 102 (catalytic triad)

Question 13

4 requirements of serine proteases

Answer 13

catalytic triad, oxyanion hole, non-specific binding site, specificity pocket

Question 14

mech/reason for catalytic triad

Answer 14

Ser forms a covalent bond with substrate => specific reaction path

His: accepts H+ from Ser, facilitates bond formation, stabilizes neg charged transition state

Asp: stabilizes pos charge of His+, increases rate ~10,000

Question 15

reason for oxyanion hole

Answer 15

Stabilizes transition state/neg charge on oxygen, forms 2 H-bonds to a neg oxygen of the substrate

Question 16

reason for non-specific binding site

Answer 16

Recognition/identity (trypsin;chymotrypsin), contributes to stabilization of transition state

Question 17

reason for specificity pocket

Answer 17

pocket of the enzyme where prefered side chain fits into, must accommodate side chain in terms of interaction and size before sessile bond

Question 18

Chymotrypsin structure

Answer 18

2 domains, Each domain: anti-parallel B -barrel, six B-strands, 1-4 is a greek key motif, 5-6 is a B antiparallel hairpin

Question 19

Chymotrypsin substrate specificity pocket

Answer 19

prefers aromatics, 189 position is Ser, makes pocket HPhob

Tooze 213

Question 20

Trypsin substrate specificity pocket

Answer 20

prefers R or K (pos charged side chains), 189 position is Asp (neg charge attracts pos charge)

(Tooze 213)

Question 21

Elastase substrate specificity pocket

Answer 21

prefers small uncharged side chains, 226 and 216 positions are Thr and Val, pocket blocked

(Tooze 213)

Question 22

Chymptrypsin active site

Answer 22

formed by 2 loop regions from each domain (3-4 and 5-6), His 57 in dom 1/loop 3-4, Ser 195 in dom 2/loop 3-4, Asp 102 in dom 1/loop 5-6; together they make catalytic triad

Question 23

What stabilizes Chymptrypsin’s active site?

Answer 23

stable scaffolding provided by B barrel

Question 24

Bacterial Subtilisin structure

Answer 24

4 A helices (2 on each side of the parallel B sheet) surrounding 5 parallel B-strands

Question 25

Bacterial Subtilisin active site

Answer 25

C-end of the central B-strands, catalytic triad

Question 26

What is Carboxypeptidase?

Answer 26

catalysis by induced electronic strain on substrate

Question 27

Zn2+ Protease

Answer 27

Glu 270 directly attacks the carbonyl carbon of the
scissle bond to form a “covalent mixed-anhydride intermediate”
Zn2+ binding -> polarizes the carbonyl
“environment”, non-polar, induced dipole
Facilitates hydrolysis by water

Question 28

Describe specificity pocket positions

Answer 28

189 is at the bottom of the pocket, 226 and 216 line the sides