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Proteins > Enzymes > Flashcards

Enzymes Flashcards

1
Q

what is delta g equal to

A

RTln(Keq)

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2
Q

what is Keq equal to

A

[X]/[A][B]

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3
Q

why is the transition state the least populated state

A

it’s the highest energy state

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4
Q

what is the equation to describe reaction rate

A

reaction rate= K’e(-deltaG/RT)[A][B]

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5
Q

what happens to reaction rate as the energy barrier gets lower

A

it increases exponentially

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6
Q

what is the effect of a catalyst

A

it reduces the energy of the transition state

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7
Q

how do you determine rate enhancement

A

work out the ratio of the rate with the catalyst and rate without the catalyst

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8
Q

what is the barrier for a reaction

A

delta Gcat/uncat

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9
Q

how is obtaining a delta G of lower energy when catalyzed achieved

A

by stabilizing the transition state

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10
Q

how must the enzyme bind the transition state in order for catalysis to occur?

A

more tightly than the substrate

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11
Q

the more tightly bound to the transition state…

A

… the greater the rate

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12
Q

what are transition state analogues

A

competitive inhibitors which resemble the TS rather than the substrate

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13
Q

what are two inhibitors of proline racemase

A

pyrrole-2-carboxylate and delta-1-pyrolline-2-carboxylate

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14
Q

what are the 5 main factors which lead to rate enhancement

A 
proximity and orientation
general acid/base catalysis
covalent catalysis
strain
cofactors
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15
Q

how does close proximity of 2 enzyme substrates increase rate

A

it increases effective concentration

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16
Q

how can pKa values be modified

A

by positioning certain residues next to others in the actve site it can extend their pKa eg lysine lysine pairs

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17
Q

for protonation of a substrate to occur- what properties should the acctive site group have in order to donate the proton

A

it should be protonated at physiological pH with a pKa just above that

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18
Q

what happens to the substrate during general base catalysis

A

a catalytic residue deprotonates water which then attacks the substrate hence deprotonating it

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19
Q

what allows histidine to act as both an acid and base

A

“perfect” pKa

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20
Q

what properties of enzyme active sites make nucleophilic catalysis so effective

A

precise orientation of nucleophile

exclusion of water

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21
Q

which amino acids have nucleophilic side chains

A

ser, thr, cys, asp, glu, lys, his, tyr

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22
Q

what is the best nucleophile available to enzymes

A

the thiol group of cysteine

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23
Q

how does strain help catalysis

A

strain can cause the substrate to resemble the TS so the energy is reduced

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24
Q

what are common single oxidation state metal ions which act as cofactors

A

Mg2+, Ca2+, Zn2+

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25
Q

what is the definition of a lewis acid

A

electron pair acceptor

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26
Q

how do metal ions improve catalysis

A

they stabalise the negative charge

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27
Q

what are common metal ion cofactors with variable oxidation states

A

nickel, manganese, iron

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28
Q

how do we obtain many organic cofactors

A

from vitamins from the diet

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29
Q

give 7 examples of common organic cofactors

A

TPP, FMN, FAD, NAD, CoA, PLP and Biotin

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30
Q

what is the role of TPP

A

helps in catalysis of the dearboxylation of alpha-keto acids

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31
Q

how does TPP stabalise the intermediate in the decarboxylation of alpha-keto acids

A

the deprotonated carbon form, known as the ylid form, allows the N of the ring to act as an electron sink, stabalising the the intermediate

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32
Q

what type of enzymes is biotin found as a cofactor

A

carboxylases

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33
Q

what is the michaelis menten equation

A

V0=Vmax[S]/Km + [S]

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34
Q

what is the basic steps of an ordered sequential Bi-Bi mechanism

A

E+A–>EA + B–> EAB-EPQ–> EQ (+P)–> E (+Q)

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35
Q

what are the basic steps of a random bi-bi mechanism

A

E + A/B–> EA/B +A/B–> EAB-EPQ–> EP/Q + P/Q–> E +P/Q

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36
Q

which two mechanisms involve a ternary complex

A

ordered sequential bi-bi and random bi-bi

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37
Q

what is the basic outline of a bi-bi ping-pong mechanism

A

E +A–> EA-E’P–> E’ (+P) +B–> E’B-EQ–> E (+Q)

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38
Q

what is the name of the fourth type of enzyme mechanism

A

theorell chance mechanism

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39
Q

what sort of enzymes commonly use the ordered sequential bi bi mechanism

A

NAD dehydrogenases

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40
Q

which sort of enzymes often use bi bi ping pong mechanism

A

pyridoxyl phosphate dependant transaminases

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41
Q

what enzyme is one of the few to use the theorell chance mechanism

A

horse liver alcohol dehydrogenase

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42
Q

what is the rate equation for ordered sequential mechanisms

A

V=Vmax[A][B]/KA’KB + KA[B] +KB[A] +[A][B]

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43
Q

What is the rate equation for ping pong mechanism

A

V=Vmax[A][B]/ KA[B] + KB[A] + [A][B]

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44
Q

what are each of the parameters of the rate equation?

A 
V=rate
[A]= conc of substrate A
[B]= conc of substrate B
KA= Km of A
KB= Km of B
Vmax= max rate of reaction when saturated with both substrates
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45
Q

a large Ka…

A

small affinity for substrate A

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46
Q

how do we measure the parameters of the rate equation

A

vary substrate concentration and measure rate at various [A] and [B] to create a 25 point matrix

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47
Q

what is the name of a graph with 1/V against 1/[A]

A

primary plot

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48
Q

what is plotted on a secondary plot

A

the slope of each line of the primary plot against 1/[B] or the intercept of the primary plot against 1/[B]

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49
Q

what does the Y intercept of a graph of the slope of the primary plot against 1/[B] tell us

A

KA/Vmax

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50
Q

what does the slope of a graph of the slope of the primary plot against 1/[B] tell us

A

KA’KB/Vmax

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51
Q

what does the Y intercept of a graph of the intercept of the primary plot against 1/[B] tell us

A

1/Vmax

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52
Q

what does the slope of a graph of the intercept of the primary plot against 1/[B] tell us

A

KB/Vmax

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53
Q

what do the lines on a primary plot for the ping pong mechanism look like?

A

parallel

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54
Q

what mechanism must be followed if a partial reaction is observed and why

A

ping pong because if P is present then e must have reacted with A in the absence of B

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55
Q

what does evidence of a modified enzyme, E’, show

A

ping pong mechanism

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56
Q

how can you determine which is substrate A and which is B in ping pong mechanisms

A

look for a partial reaction. whichever produces the partial reaction must be A

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57
Q

how can you determine which is substrate A and which is B in ordered sequential mechanisms

A

equilibrium dialysis- if ES complex is formed then the [S] will be greater inside the bag than out
surface plasma resonance

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58
Q

how can we determine if its ordered squential or random bi bi

A

set up two reactions one containing radioactive A and one containing radioactive B, both with the enzyme. if both bind its random, if only one binds its ordered.

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59
Q

what is the basis for product inhibition patterns

A

products typically act as inhibitors since they are formed in the active site

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60
Q

as well as varying A + B and P + Q, what other conditions can be varied to give 8 possible combinations in product inhibition patterns

A

saturated or non saturated

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61
Q

what is rule 1 of product inhibition patterns

A

the Y intercept of a reciprical plot is affected by a compound which associates reversibly with an enzyme form other than the one with which the variable substrate binds

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62
Q

what is rule 2 of product inhibition patterns

A

the slope of a reciprical plot is affected by a compound which associates with an enzyme form that is the same as or is connected by a series of reversible steps to the enzyme form to which the variable substrate combines

63
Q

under what circumstances are steps not considered reversible, concerning the rules of product inhibition patterns

A

when saturation with a substrate will block the reversible step
or when the other product is involved amd we consder that its concentration is 0.

64
Q

if the km changes but vmax remains the same on a lineweaver burke plot, what sort of inhibition is demonstrated

A

competitive

65
Q

if the vmax and km changes on a lineweaver burke plot, what sort of inhibition is demonstrated

A

uncompetitive

66
Q

if the vmax changes but km remains the same on a lineweaver burke plot, what sort of inhibition is demonstrated

A

non-competitive

67
Q

what is the mr of lactate dehydrogenase

A

140kDa

68
Q

what are the two subunits which lactate dehydrogenase is made up of called

A

alpha and beta

69
Q

how many subutits does LDH have

A

4 (tetrameric)

70
Q

where is alpha4 ldh usually found

A

heart (H4)

71
Q

where is B4 ldh usually found

A

skeletal muscle

72
Q

how can ldh be used to determine whether or not someone has had a heart attack or pulled a muscle

A

LDH in blood- a4=heart attack, b4=pulled muscle

73
Q

what sort of kinetic mechanism does ldh follow

A

ordered sequential

74
Q

why are competitive inhibitors good for solving x ray rystallography

A

they bind to the same site as substrate

75
Q

what is oxalate a comp inhibitor with respect to

A

pyruvate

76
Q

what is oxalate a comp inhibitor w respect to

A

lactate

77
Q

what is the reaction catalysed by LDH

A

NAD+ + lactate –> NADH + Pyruvate

78
Q

how can we measure the first few ms of a reaction

A

pre-steady state kinetics using continuous flow spectrophotometry or stopped flow spec

79
Q

how are syringes driven in continuous/stopped flow kinetics

A

releasing a N cylinder

80
Q

why is continuous flow spec used more commonly nowadays

A

can view whole time period as opposed to just one point and the need for large amounts of E and S is no longer an economical problem due to over expression and genetics

81
Q

what are the first few ms of a react known as

A

burst phase

82
Q

what did cont flow kinetics show about ldh

A

NADH production, release and pyruvate release is quick

83
Q

what further techniques were used to determine slow step

A

affinity labels and general labels

84
Q

what do affinity labels do

A

only label a protein if it shows affinity for it

85
Q

how does bromopyruvate bind ldh

A

its similar to pyruvate so binds in a similar wayin active site

86
Q

what is the result of ldh binding bromorpyruvate

A

nucleophilic residue important for catalysis attacks Br resulting in protein being modified, resulting in denaturation of ldh

87
Q

how is Br detected

A

its radiolabelled

88
Q

what occurs in the process after bromopyruvate binds the protein

A

protein unfolded in urea
treated with proteases resulting in fragments, one of which will be radioactive
fragments separated
radioactive peptide purified and sequenced
determine position of residue

89
Q

which residue does bromopyruvate modify

A

His195

90
Q

what does binding of br to his195 tell you about the residue

A

its in the active site, positioned near substrate and likely to be catalytic

91
Q

what are general labels

A

bind to a particular type of aa but don’t necessarily show affinity

92
Q

what is DEPC

A

a histidine directed label- will modify potentially any his residues

93
Q

what can be determined by loss of activity after depc treatment

A

there must be at least one his involved in catalysis

94
Q

which his residue in ldh is modified by depc

A

his 195

95
Q

what residues does phenylglyoxal modify

A

arg

96
Q

what does modification with phenylglyoxal show

A

1 arg per subunit is modified

97
Q

why can the residue number of the arginines modified not be determined by the same method as affinity labellng

A

not stable enough

98
Q

how can it be shown that phenylglyoxal modifies a arg residue in the active site

A

activity against time assay showing that rate decreases with addition of phenylglyoxal to enzyme

99
Q

why does rate remain the same with ldh + phenylglyoxal + lactate + nad

A

substrate protection

100
Q

what does substrate protection determine

A

residue in active site

101
Q

what residues does NEM bind

A

cysteine

102
Q

what is the result of NEM + LDH

A

inactivation and substrate protection

103
Q

which residue is modified by nem

A

cys165

104
Q

which molecule in the LDH reaction demonstrates chirality

A

nad+

105
Q

why can only one specific H be removed

A

enzyme precents rotaion- stereochemistry

106
Q

which sort of dehydrogenates always remove Ha

A

A site dehydrogenases

107
Q

what sort of dehydrogenase is ldh

A

a site

108
Q

how can we determine whether an enzyme is A or B side specific

A

label the Hs radioactively to give info on the orientation in the active site

109
Q

how does orientation of nadh help us determine mechanism of ldh

A

tells us pyruvate must be on the other side of the active site

110
Q

what is the n terminal domain of the active site made up of

A

6 stranded b sheet with 4 alpha helices

111
Q

what does the n terminal bind

A

NAD/H

112
Q

what is the c terminal made up of

A

2x 3 stranded anti parallel b sheets

113
Q

what does the c terminal bind

A

substrate- lactate/pyruvate

114
Q

what is the n terminal of dehydrogenates known as

A

rossman fold

115
Q

what is the structure of the rossman fold

A

bababa diamer

116
Q

what does the rossman fold form in order to bind nad

A

a groove

117
Q

what causes ldh to be a side specific

A

lysine h bonds with o preventing roatation

118
Q

which two structures whow a large conformational change

A

E and E-NADH

119
Q

where is the flexible loop found

A

residues 98-120

120
Q

what happens to the loop upon binding of nadh

A

it closes

121
Q

what residue is in middle of loop

A

arg 109

122
Q

what is the purpose of loop closure

A

expel water from active site

123
Q

why was site directed mutagenesis carried out

A

to test importance of specific residues such as arg 109

124
Q

what did the mutation of arg109 to glutamine (R109Q) cause/show

A

Kcat 400x lower= R involved in catalysis

125
Q

how is arg109 involved in catalysis

A

stabilises the TS due to its +ve carge

126
Q

why are proteins inherently fluorescent

A

tryptophan residues

127
Q

what effect does changing Trp residues fro Y residues have

A

still active but not fluorescent

128
Q

what mutation was added to the mutant containing Y in place of W residues

A

the G106 residue was mutated for a tryptophan, giving fluorescence in middle of loop

129
Q

why would fluorescence change depending on open or closed state of loop

A

fluorescence sensitive to environment

130
Q

what was used to determine that oxamate triggers loop closure and the rate of closure

A

pre steady state kinetics of the E-NADH complex with oxamate

131
Q

what was the rate of loop closure and what did this show

A

125/s

slow step in the reaction

132
Q

what caused the apparent substrate protection upon the addition of NEM, now suggesting cya 165 isn’t in fact involved in catalysis

A

loop closure prevented cys165 from being accessible to NEM

133
Q

what is the michaelis menten equation with relation to Kcat

A

V=Kcat[E][S]/km[S]

134
Q

what is the equation for the specificity constant

A

kcat/km

135
Q

how do you find the relative rate of an enzyme with two diff substrates

A

ratio of kcat/km

136
Q

what did scientists try to change the specificty off ldh to

A

oxaloacetate and malate

137
Q

what did they not want to change in the experiment to change the specificity of ldh

A

anything to do wit nad/h binding
binding of carboxalate groups
mechanism
loop closure

138
Q

what is the diff between pyruvate and oxaloacetate

A

bigger and more negative

139
Q

what were the focus features of the experiment

A

charge balance
substrate size vs active site volume
direct electrostatic complementarity

140
Q

what was the overall charge in the wt reaction compared to mutant and what did this mean for the experiment

A

0 and -1

need to change active site to be less negative

141
Q

what 2 mutations were introduce to make active site less negative and what were the results

A

D197N- less with P but same with O

E107Q- lees activity with P, more with O but negligible

142
Q

was E or D a buried residue

A

D- larger effect with exposed residue

143
Q

how was the pocket made larger and what was result

A

thr246gly

greatly decresed w P, slightly increased with O

144
Q

which mutation produced a huge switch in specificity

A

Gln102Arg

145
Q

what is glutathione made up of

A

g-glutamate, cysteine and glycine

146
Q

what is the oxidised version of glutathione called

A

GSSG

147
Q

what reaction does glutathione reductase catalyse

A

GSSG + NADPH + H –> 2GSH + NADP

148
Q

which family of proteins does glutathione reductase belong to

A

flavoprotein disulphide oxidoreductases

149
Q

how many subunits aew in glutathione reductase

A

2 (dimeric)

150
Q

what was the aim of the protein engineering experiments with glutathione reductase

A

change specificity from nadph to nadh

151
Q

what is the active site of glutathione reductase divided by

A

fad

152
Q

what do we not want to change in glutathione reductase

A

anything to do with glutathione binding

anything involved in binding fad

153
Q

what do we want to change in glutathione reductase

A

how it binds nadph0
nad smaller and less negative
focus on very positive bnding pocket

154
Q

how many mutations were required to change specificity

A

7

Proteins (4 decks)

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