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bm210 biochemistry > B2 mechanisms of enzyme catalysis > Flashcards

B2 mechanisms of enzyme catalysis Flashcards

1
Q

what is the essence of catalysis

A

the specific stabilisation of the transition state

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2
Q

what do catalysts do

A

-lower the activation energy
-accelerator of a chemical reaction
-increase the rate of rxn
-is not consumed in the rxn
-does not affect the eqm

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3
Q

how is the active site of an enzyme formed

A

folding of protein brings side chains of various aa that may be far apart in the primary seq into close juxtaposition

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4
Q

how many steps are in the enzyme-catalysed reaction

A

3
enz+S– enz-S–enz-P–enz+p

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5
Q

what allows the reaction to occur in regards to active site and substrate

A

positioning of the substrate mols in the most favourable relative orientation for the rxn to occur

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6
Q

the active site is perfectly ??? to the transition state

A

complementary

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7
Q

what do the aa side chains of the active site do to the electron distribution of the transition state

A

stabilise

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8
Q

the substrate is ??? on binding to the active site

A

strained

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9
Q

enzymes do what to the activation energy and reaction rate

A

lowers the activation energy
increases the reaction rate

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10
Q

what are the non covalent interactions between the substrate and the aa side chains of the enzyme

A

-acidic groups (Asp, Glu)= ionic bonds
-basic groups (Lys, His, Arg)= ionic bonds
-hydrophillic ints woth -OH or alc groups (Ser, Thr, Tyr)
-hydrophilic ints with -SH or thiol groups (Cys)
-hydrophilic ints with amide groups (Asm, Gln)
-aromatic ints (Phe, Tyr, Trp)
-hydrophobic ints (Ala, Leu, Lle, Val, Met)

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11
Q

how do reactive groups at the catalytic site surface catalyse the rxn by

A

-donating or withdrawing electrons
-stabilising or generating free radical intermediates
-forming temporary covalent bonds
(transition state intermediate)

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12
Q

what are cofactors

A

non protein molecules in addition to enzymes

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13
Q

3 examples of cofactors

A

-metal group (hexokinase Mg2+)
-prosthetic group
covalently bound organic mol (heme, lipoic acid)
-coenzyme
tightly but not covalently bound organic mol (NAD)

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14
Q

name of an enzyme protein WITH prosthetic group/coenzyme and is it catalytically active

A

holo-enzyme
catalytically active

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15
Q

name of an enzyme protein WITHOUT prosthetic group/coenzyme and is it catalytically active

A

apoenzyme
catalytically inactive

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16
Q

what does specificity mean

A

enzymes catalyse only one specific rxn

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17
Q

oxidoreductases (general)

A

oxidation and reduction reactions

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18
Q

dehydrogenases

A

addition or removal of H

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19
Q

oxidases

A

2 electron transfer of O2 forming H2O2
2 electron transer to 1/2 O2 forming H2O

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20
Q

oxygenases

A

incorporate O2 into product

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21
Q

hydroxlases

A

incorporate 1/2 O2 into product as -OH and form H2O

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22
Q

peroxidases

A

use as H2O2 as oxygen donor, forming H2O

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23
Q

transferases (general)

A

transfer a chemical group from one substrate to another

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24
Q

kinases

A

transfer phosphate from ATP onto substrate

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25
Q

hydrolases

A

hydrolysis of C-O, C-N, O-P, and C-S bonds

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26
Q

examples of hydrolases

A

esterases, proteases, phosphatases, thioesterases

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27
Q

lyases

A

addition across a carbon-carbon double bond

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28
Q

examples of lyases

A

dehydratases, hydratases, decarboxylases

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29
Q

isomerases

A

intramolecular rearrangements

30
Q

synthetases

A

formation of bonds between two substrates
frequently linked to utilisation of ATP

31
Q

units of catalytic/enzyme activity

A

number of micromoles (umol) of substrate converted per minute under standard optimised conditions at 30 degrees
1 enzyme unit (EU)= 1umol min-1

32
Q

what is specific activity

A

activity of an enzyme per milligram (mg) of total protein in the enzyme prep
(expressed in umol min-1mg-1)

33
Q

what does specific activity give a measurement of

A

purity of the enzyme

34
Q

why is the rxn rate hyperbolic

A

accumulation of product
depletion of substrate
denaturation of enzyme

35
Q

requirements of measuring the rxn rate

A

measured at fixed enzyme conc
defined temp and pH

36
Q

for meaningful quantitative assays of enzyme activity it is necessary to ensure what

A

that initial velocities (V0; rxn rates) are measured

37
Q

5 factors that affect enzyme activity

A

-pH
-temperature
-concentration of enzyme
-concentration of substrate
-covalent modification of enzyme

38
Q

what is pH and its parameters

A

a measure of the acidity of alkalinity of a solution
acidic < 7.00 < basic
neutral pH = 7.00

39
Q

what of the aa side chains depends on the pH of the sol

A

ionisation state

40
Q

what is dependent on the pH of an enzyme catalysed rxn

A

binding of the substrate and catalysis

41
Q

do chemical reactions proceed faster or slower at higher temps and why

A

faster
mols move faster, greater chance to strike
electrons gain activation energy easier

42
Q

what happens when an enzyme is denatured

A

loss of H bonding
unfolding
precipitation
loss of activity

43
Q

the effect of varying the amount of enzyme

A

predictable linear increase in product formation with increasing amount of enzyme

44
Q

what does the michaelis-menten eqn describe

A

the dependence of rate of rxn on concentration of substrate at steady state (ES formation balanced by its removal) and vast molar excess of substrate over enzyme [S]»[E]

45
Q

what do the following in the michaelis menten eqn mean
v
Vmax
[S]
Km

A

v rate of rxn
Vmax maximal rate of rxn
[S] conc of substrate
Km Michaelis constant

46
Q

what is the michaelis menten eqn

A

v= Vmax[S]/Km + [S]

47
Q

what does high Km and low Kn correspond to in terms of substrate

A

high = low affinity
low = high affinity

48
Q

enzymes with low Km compared with the conc of substrate [S] in the cell act at their ?

A

max rate

49
Q

do modest changed in the conc of substrate [S] have an effect on the rate of rxn

A

no

50
Q

effect of rate of rxn where enzymes with a high Km and a small change in [S] conc

A

large change

51
Q

experimental determination of Km and Vmax

A

-incubation of the enzyme under optimal conditions for a short time
(assuming no change on conc of [S] or [product] is negligible compared with [S]
-using range of [S] concs
-plotting double reciprocal graph of rxn rate over [S] conc
-extrapolating back from experimental points to determine intercepts

52
Q

what is the plot given to the double reciprocal plot

A

lineweaver-burk
1/rate
1/Vmax
-1/Km
1/[substrate]

53
Q

what occurs in a sequential rxn
(enzyme with 2 substrates)

A

each substrate binds in turn

54
Q

what is a ternary complex
and what do the lines look like on LB graph

A

complex containing three diff mols A-Enz-B
converging

55
Q

dihydrofolate reductase enzyme pathway

A

dihydrofolate+NADPH+H+—-tetrahydrofolate+NADP+

56
Q

ping pong rxn
(enzymes with 2 substrates)
and what do the lines look like on LB graph

A

one substrate reacts, and modifies enzyme
then second substrate reacts with modified enzyme
parallel lines

57
Q

what is an allosteric enzyme and where are the often found

A

contain binding sites other “allo” than substrate binding sites
often in multi subunit complex
more than one active site in complex

58
Q

what does the binding of a substrate to an allosteric enzyme lead to

A

binding to active site of first subunit leads to change in conformation facilitating binding of substrate to the other active site

59
Q

what is a reversible inhibitor

A

non covalent (eqm) binding to enzyme
many are relatively unspecific

60
Q

mechanism of a reversible inhibitor

A

blocking substrate binding or hindering catalytic steps

61
Q

what are irreversible inhibitors

A

inactivators
bind to enzyme covalently (suicide inhibitor)
many are substrate analogues
undergo part of rxn
transition state covalent intermediate does not break down

62
Q

what is a competitive inhibitor

A

competes with substrate for binding at the active site
is a function of the relative affinities of the substrate and the inhibitor for binding the enzyme
inhibition is a function of relative conc of substrate and inhibitor

63
Q

effect of competitive reversible inhibition on michaelis menten and LB

A

Vmax is unchanged, Km increased
if enough substrate added, can overcome inhibitor

64
Q

what are mixed inhibitors

A

they do not bind to the active site
inhibitor can bind prior to substrate or to the enzyme-substrate complex

65
Q

mixed inhibitors distort the substrate binding site which affects

A

-apparent substrate affinity
-catalytic turnover (slowing catalysis)

66
Q

effect of mixed inhibitors on Km and Vmax

A

either increase or decrease Km
decrease Vmax

67
Q

binding position and competition of non competitive inhibitors

A

binds to the enzyme at a position separate from the active site
no competition for binding with the substrate

68
Q

what happens to the affinity and rate of rxn with non competitive inhibitors

A

apparent affinity for the substrate is unchanged, but the rate of rxn is slowed

69
Q

effect of non competitive inhibition on MM eqn
and effect of adding more [S]

A

Km is unchanged, Vmax is decreased
adding more substrate has no effect on the rate of rxn

70
Q

effect of allosteric inhibitors on Km and apparent affinity of enzyme

A

increase the Km and lower to apparent affinity of enzyme for its substrate

bm210 biochemistry (19 decks)

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