10. Enzyme mechanisms

Question 1

what does an enzymes catalytic activity depend on

Answer 1

their native conformation

Question 2

T or F: an enzymes catalytic activity can function when the enzyme unfolds

Answer 2

false; catalytic activity depends on their native conformation and will usually be lost if any degree of unfolding happens

Question 3

where on the enzyme does the reaction take place

Answer 3

the active site

Question 4

T or F: enzymes often require cofactors/coenzymes

Answer 4

true

Question 5

what is a cofactor

Answer 5

a metal ion

Question 6

what is a coenzyme

Answer 6

one or more organic/metalloorganic complexes, usually derived from vitamins

Question 7

what do the coenzymes carry

Answer 7

a specific functional group

Question 8

when the cofactor/coenzyme is tightly bound to the enzyme, what are they called

Answer 8

prosthetic group

Question 9

what is free energy

Answer 9

the energy available to do work

Question 10

describe the free energy in an exergonic reaction

Answer 10

free energy decreases

Question 11

what does a reaction coordinate diagram look like

Answer 11

free energy vs progress of the reaction. There is a peak as S turns into P. There are troughs representing ES and EP

Question 12

T or F: if a reaction is exergonic, it will be fast

Answer 12

false; just because something is spontaneous doesn’t mean it will be fast

Question 13

what is a transition state

Answer 13

it’s the tall peak on a graph, and it represents an energy barrier as we go from S to P

Question 14

what is the activation energy (Ea)

Answer 14

the difference between transition state and ground state of S

Question 15

T or F; at the transition state, progress towards S or P is equally likely

Answer 15

true

Question 16

explain why progress towards S or P is equally likely at the transition state

Answer 16

aligning groups and bond breakage have occurred to a point where decay to the products or decay to the substrates have the same odds

Question 17

what does ∆Gǂ measure

Answer 17

∆Gǂ is the activation energy for the reaction. It measures the difference between the energy levels of the ground and transition states

Question 18

what does a high activation energy mean

Answer 18

slower reaction

Question 19

in regards to ∆Gǂ, what is the role of the enzyme

Answer 19

to decrease ∆Gǂ by lowering the transition state

Question 20

why is a lock and key a bad analogy for substrates and enzymes

Answer 20

if the substrate perfectly fit into the enzyme would stabilize the substrate, and a product would not form. It would increase the activation energy needed to form P

Question 21

what is binding energy (∆GB)

Answer 21

the free energy released from ES interactions in the transition state. It is the difference between the cat. and uncat. reactions

Question 22

T or F: if there are multiple reaction intermediates, overcoming each barrier may require a dif. activation energy

Answer 22

true

Question 23

which step is the rate limiting step

Answer 23

the step with the highest activation energy (has the highest peak)

Question 24

what are the 6 categories of enzymes

Answer 24

oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases

Question 25

describe oxidoreductases

Answer 25

catalyze redox reactions, most in this group are dehydrogenases, oxidases, peroxidases, oxygenases, or reductases

Question 26

describe transferases

Answer 26

catalyze group transfer reactions (ie kinases)

Question 27

describe hydrolases

Answer 27

catalyze hydrolysis, are special class of transferases with water serving as the acceptor of the group transferred

Question 28

describe lyases

Answer 28

catalyze lysis of a substrate which generates a double bond or a ring

Question 29

describe isomerases

Answer 29

catalyze structural changes within a single molecule to produce an isomer

Question 30

describe ligases

Answer 30

catalyze ligation or joining of two substrates (usually linked to the breakage of a pyrophosphate bond)

Question 31

what type of amino acids is the active site lined with

Answer 31

hydrophobic ones

Question 32

why is the active site lined with hydrophobic amino acids

Answer 32

they help stabilize substrates through hydrophobic interactions

Question 33

which residues in an enzyme are directly involved in the catalysis (there's 5)

Answer 33

His, Asp, Arg, Glu, and Lys

Question 34

what is the role of His, Asp, Arg, Glu, and Lys in catalysis

Answer 34

they function as acid, base, and nucleophiles, and they're more likely to bind and stabilize transition states and cofactors

Question 35

other than binding energy, how do enzymes promote catalysis

Answer 35

once an S is bound to E, properly positioned functional groups in E aid in the cleavage and formation of bonds to form P

Question 36

name the 3 mechanisms for catalysis

Answer 36

1. general acid base catalysis 2. covalent catalysis 3. metal ion catalysis

Question 37

describe general acid-base catalysis

Answer 37

it's a proton transfer mediated by weak acids and bases (not water). These acids/bases are amino acid residues within the active site

Question 38

describe covalent catalysis

Answer 38

a transient covalent bond is formed between E and S. The side chain of the involved amino acid in the active site is either a nucleophile or an electrophile

Question 39

describe metal ion catalysis

Answer 39

metals can either be tightly bound as cofactors or taken up from the solution along with the substrate. Ionic interactions between the metal and S can help orient S for the reaction

Question 40

what are serine proteases

Answer 40

a class of enzymes that cleave peptide bonds. They're characterized by the presence of catalytic serine residues within their active sites

Question 41

give 3 examples of serine proteases

Answer 41

chymotrypsin, trypsin, and elastase

Question 42

where are the serine proteases synthesized

Answer 42

in the pancreas

Question 43

in what form are the serine proteases stored

Answer 43

stored as zymogens

Question 44

list the names of the serine proteases when they're in their zymogen form

Answer 44

chymotrypsinogen, trypsinogen, and proelastase

Question 45

why are the serine proteases stored as zymogens

Answer 45

so they're not degrading protein when they're not supposed to

Question 46

T or F: trypsin, chymotrypsin, and elastase are homologs

Answer 46

true

Question 47

explain how the serine proteases are homologs

Answer 47

all 3 have a two-lobed structure with the active site in a cleft between the two domains. The positions of the catalytically active side chains (Ser, His, and Asp) are almost identical

Question 48

describe how chymotrypsin's active site differs from the others

Answer 48

it catalyzes cleavage of peptides on the carbonyl side of uncharged residues with aromatic or bulky hydrophobic side chains

Question 49

describe how trypsin's active site differs from the others

Answer 49

catalyzes cleavage of peptides on the carbonyl side of Arg and Lys

Question 50

describe how elastase's active site differs from the others

Answer 50

catalyzes the cleavage of elastin, a fibrous protein rich in Gly and Ala residues

Question 51

describe the structure of chymotrypsin

Answer 51

3 polypeptide chains linked by disulfide bonds. Has a hydrophobic pocket where the bulky amino acid side chain of S will bind

Question 52

what are the 2 reaction phases of chymotrypsin

Answer 52

acylation phase | deacylation phase

Question 53

what is the acylation phase

Answer 53

peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme (specifically serine)

Question 54

what is the deacylation phase

Answer 54

the ester linkage is hydrolyzed and a non acylated enzyme is regenerated

Question 55

what are the 3 catalytic residues of chymotrypsin

Answer 55

His57, Asp102, Ser195

Question 56

what do the 3 catalytic residues of chymotrypsin form

Answer 56

a hydrogen bonding network called the catalytic triad

Question 57

describe the location of the H bonds within the catalytic triad

Answer 57

Asp with His, which is bonded to Ser

Question 58

T or F: Gly is in the catalytic triad

Answer 58

false

Question 59

since Gly isn't in the catalytic triad, what is it's role

Answer 59

it has a backbone N that contributes to an H bond to the tetrahedral intermediate

Question 60

what nucleophile starts the chymotrypsin mechanism

Answer 60

the deprotonated oxygen of the serine OH group

Question 61

where will the substrate be cleaved

Answer 61

on the carboxyl side of the residue in the middle

Question 62

what is an oxyanion hole

Answer 62

an oxyanion is an oxygen anion (O-) so O- will go in the hole

Question 63

describe what happens when a substrate binds to chymotrypsin

Answer 63

causes a conf. change, bringing Asp and His closer together, compressing the H bond between them. Now, His REALLY wants a proton, and it will get it from the OH group of Ser. Also, the side chain of the residue adjacent to the pep bond nestled in a hydrophobic pocket

Question 64

what effect does deprotonation have on Ser

Answer 64

it makes it a really strong nucleophile

Question 65

see notes for the rest of chymotrypsin mechanism

Question 66

why do enzymes have a pH range where their activity is maximal

Answer 66

as pH varies, dif. R groups may change their ionization status

Question 67

what pH is chymotrypsin's peak velocity around

Answer 67

8

Question 68

T or F: both kcat and Km will be affected by pH changes

Answer 68

true

Question 69

what affect will a protonated His have on kcat

Answer 69

it will lower the kcat because when it's protonated it no longer needs to steal protons from Ser

Question 70

Km is ___ at low pH values

Answer 70

low

Question 71

what changes as Km increases as pH increases

Answer 71

polypeptide B of chymotrypsin has Ile16 at the N term, and it normally forms a salt bridge with Asp194 when charged. The salt bridge stabilized the active site, so when you protonate you lose the bridge and therefore stabilization (this collapses the hydrophobic pocket)