Enzymes Flashcards
1
Q
what is delta g equal to
A
RTln(Keq)
2
Q
what is Keq equal to
A
[X]/[A][B]
3
Q
why is the transition state the least populated state
A
it’s the highest energy state
4
Q
what is the equation to describe reaction rate
A
reaction rate= K’e(-deltaG/RT)[A][B]
5
Q
what happens to reaction rate as the energy barrier gets lower
A
it increases exponentially
6
Q
what is the effect of a catalyst
A
it reduces the energy of the transition state
7
Q
how do you determine rate enhancement
A
work out the ratio of the rate with the catalyst and rate without the catalyst
8
Q
what is the barrier for a reaction
A
delta Gcat/uncat
9
Q
how is obtaining a delta G of lower energy when catalyzed achieved
A
by stabilizing the transition state
10
Q
how must the enzyme bind the transition state in order for catalysis to occur?
A
more tightly than the substrate
11
Q
the more tightly bound to the transition state…
A
… the greater the rate
12
Q
what are transition state analogues
A
competitive inhibitors which resemble the TS rather than the substrate
13
Q
what are two inhibitors of proline racemase
A
pyrrole-2-carboxylate and delta-1-pyrolline-2-carboxylate
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
15
Q
how does close proximity of 2 enzyme substrates increase rate
A
it increases effective concentration
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
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
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
19
Q
what allows histidine to act as both an acid and base
A
“perfect” pKa
20
Q
what properties of enzyme active sites make nucleophilic catalysis so effective
A
precise orientation of nucleophile
exclusion of water
21
Q
which amino acids have nucleophilic side chains
A
ser, thr, cys, asp, glu, lys, his, tyr
22
Q
what is the best nucleophile available to enzymes
A
the thiol group of cysteine
23
Q
how does strain help catalysis
A
strain can cause the substrate to resemble the TS so the energy is reduced
24
Q
what are common single oxidation state metal ions which act as cofactors
A
Mg2+, Ca2+, Zn2+
25
what is the definition of a lewis acid
electron pair acceptor
26
how do metal ions improve catalysis
they stabalise the negative charge
27
what are common metal ion cofactors with variable oxidation states
nickel, manganese, iron
28
how do we obtain many organic cofactors
from vitamins from the diet
29
give 7 examples of common organic cofactors
TPP, FMN, FAD, NAD, CoA, PLP and Biotin
30
what is the role of TPP
helps in catalysis of the dearboxylation of alpha-keto acids
31
how does TPP stabalise the intermediate in the decarboxylation of alpha-keto acids
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
32
what type of enzymes is biotin found as a cofactor
carboxylases
33
what is the michaelis menten equation
V0=Vmax[S]/Km + [S]
34
what is the basic steps of an ordered sequential Bi-Bi mechanism
E+A-->EA + B--> EAB-EPQ--> EQ (+P)--> E (+Q)
35
what are the basic steps of a random bi-bi mechanism
E + A/B--> EA/B +A/B--> EAB-EPQ--> EP/Q + P/Q--> E +P/Q
36
which two mechanisms involve a ternary complex
ordered sequential bi-bi and random bi-bi
37
what is the basic outline of a bi-bi ping-pong mechanism
E +A--> EA-E'P--> E' (+P) +B--> E'B-EQ--> E (+Q)
38
what is the name of the fourth type of enzyme mechanism
theorell chance mechanism
39
what sort of enzymes commonly use the ordered sequential bi bi mechanism
NAD dehydrogenases
40
which sort of enzymes often use bi bi ping pong mechanism
pyridoxyl phosphate dependant transaminases
41
what enzyme is one of the few to use the theorell chance mechanism
horse liver alcohol dehydrogenase
42
what is the rate equation for ordered sequential mechanisms
V=Vmax[A][B]/KA'KB + KA[B] +KB[A] +[A][B]
43
What is the rate equation for ping pong mechanism
V=Vmax[A][B]/ KA[B] + KB[A] + [A][B]
44
what are each of the parameters of the rate equation?
```
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
```
45
a large Ka...
small affinity for substrate A
46
how do we measure the parameters of the rate equation
vary substrate concentration and measure rate at various [A] and [B] to create a 25 point matrix
47
what is the name of a graph with 1/V against 1/[A]
primary plot
48
what is plotted on a secondary plot
the slope of each line of the primary plot against 1/[B] or the intercept of the primary plot against 1/[B]
49
what does the Y intercept of a graph of the slope of the primary plot against 1/[B] tell us
KA/Vmax
50
what does the slope of a graph of the slope of the primary plot against 1/[B] tell us
KA'KB/Vmax
51
what does the Y intercept of a graph of the intercept of the primary plot against 1/[B] tell us
1/Vmax
52
what does the slope of a graph of the intercept of the primary plot against 1/[B] tell us
KB/Vmax
53
what do the lines on a primary plot for the ping pong mechanism look like?
parallel
54
what mechanism must be followed if a partial reaction is observed and why
ping pong because if P is present then e must have reacted with A in the absence of B
55
what does evidence of a modified enzyme, E', show
ping pong mechanism
56
how can you determine which is substrate A and which is B in ping pong mechanisms
look for a partial reaction. whichever produces the partial reaction must be A
57
how can you determine which is substrate A and which is B in ordered sequential mechanisms
equilibrium dialysis- if ES complex is formed then the [S] will be greater inside the bag than out
surface plasma resonance
58
how can we determine if its ordered squential or random bi bi
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.
59
what is the basis for product inhibition patterns
products typically act as inhibitors since they are formed in the active site
60
as well as varying A + B and P + Q, what other conditions can be varied to give 8 possible combinations in product inhibition patterns
saturated or non saturated
61
what is rule 1 of product inhibition patterns
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
62
what is rule 2 of product inhibition patterns
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
under what circumstances are steps not considered reversible, concerning the rules of product inhibition patterns
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
if the km changes but vmax remains the same on a lineweaver burke plot, what sort of inhibition is demonstrated
competitive
65
if the vmax and km changes on a lineweaver burke plot, what sort of inhibition is demonstrated
uncompetitive
66
if the vmax changes but km remains the same on a lineweaver burke plot, what sort of inhibition is demonstrated
non-competitive
67
what is the mr of lactate dehydrogenase
140kDa
68
what are the two subunits which lactate dehydrogenase is made up of called
alpha and beta
69
how many subutits does LDH have
4 (tetrameric)
70
where is alpha4 ldh usually found
heart (H4)
71
where is B4 ldh usually found
skeletal muscle
72
how can ldh be used to determine whether or not someone has had a heart attack or pulled a muscle
LDH in blood- a4=heart attack, b4=pulled muscle
73
what sort of kinetic mechanism does ldh follow
ordered sequential
74
why are competitive inhibitors good for solving x ray rystallography
they bind to the same site as substrate
75
what is oxalate a comp inhibitor with respect to
pyruvate
76
what is oxalate a comp inhibitor w respect to
lactate
77
what is the reaction catalysed by LDH
NAD+ + lactate --> NADH + Pyruvate
78
how can we measure the first few ms of a reaction
pre-steady state kinetics using continuous flow spectrophotometry or stopped flow spec
79
how are syringes driven in continuous/stopped flow kinetics
releasing a N cylinder
80
why is continuous flow spec used more commonly nowadays
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
what are the first few ms of a react known as
burst phase
82
what did cont flow kinetics show about ldh
NADH production, release and pyruvate release is quick
83
what further techniques were used to determine slow step
affinity labels and general labels
84
what do affinity labels do
only label a protein if it shows affinity for it
85
how does bromopyruvate bind ldh
its similar to pyruvate so binds in a similar wayin active site
86
what is the result of ldh binding bromorpyruvate
nucleophilic residue important for catalysis attacks Br resulting in protein being modified, resulting in denaturation of ldh
87
how is Br detected
its radiolabelled
88
what occurs in the process after bromopyruvate binds the protein
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
which residue does bromopyruvate modify
His195
90
what does binding of br to his195 tell you about the residue
its in the active site, positioned near substrate and likely to be catalytic
91
what are general labels
bind to a particular type of aa but don't necessarily show affinity
92
what is DEPC
a histidine directed label- will modify potentially any his residues
93
what can be determined by loss of activity after depc treatment
there must be at least one his involved in catalysis
94
which his residue in ldh is modified by depc
his 195
95
what residues does phenylglyoxal modify
arg
96
what does modification with phenylglyoxal show
1 arg per subunit is modified
97
why can the residue number of the arginines modified not be determined by the same method as affinity labellng
not stable enough
98
how can it be shown that phenylglyoxal modifies a arg residue in the active site
activity against time assay showing that rate decreases with addition of phenylglyoxal to enzyme
99
why does rate remain the same with ldh + phenylglyoxal + lactate + nad
substrate protection
100
what does substrate protection determine
residue in active site
101
what residues does NEM bind
cysteine
102
what is the result of NEM + LDH
inactivation and substrate protection
103
which residue is modified by nem
cys165
104
which molecule in the LDH reaction demonstrates chirality
nad+
105
why can only one specific H be removed
enzyme precents rotaion- stereochemistry
106
which sort of dehydrogenates always remove Ha
A site dehydrogenases
107
what sort of dehydrogenase is ldh
a site
108
how can we determine whether an enzyme is A or B side specific
label the Hs radioactively to give info on the orientation in the active site
109
how does orientation of nadh help us determine mechanism of ldh
tells us pyruvate must be on the other side of the active site
110
what is the n terminal domain of the active site made up of
6 stranded b sheet with 4 alpha helices
111
what does the n terminal bind
NAD/H
112
what is the c terminal made up of
2x 3 stranded anti parallel b sheets
113
what does the c terminal bind
substrate- lactate/pyruvate
114
what is the n terminal of dehydrogenates known as
rossman fold
115
what is the structure of the rossman fold
bababa diamer
116
what does the rossman fold form in order to bind nad
a groove
117
what causes ldh to be a side specific
lysine h bonds with o preventing roatation
118
which two structures whow a large conformational change
E and E-NADH
119
where is the flexible loop found
residues 98-120
120
what happens to the loop upon binding of nadh
it closes
121
what residue is in middle of loop
arg 109
122
what is the purpose of loop closure
expel water from active site
123
why was site directed mutagenesis carried out
to test importance of specific residues such as arg 109
124
what did the mutation of arg109 to glutamine (R109Q) cause/show
Kcat 400x lower= R involved in catalysis
125
how is arg109 involved in catalysis
stabilises the TS due to its +ve carge
126
why are proteins inherently fluorescent
tryptophan residues
127
what effect does changing Trp residues fro Y residues have
still active but not fluorescent
128
what mutation was added to the mutant containing Y in place of W residues
the G106 residue was mutated for a tryptophan, giving fluorescence in middle of loop
129
why would fluorescence change depending on open or closed state of loop
fluorescence sensitive to environment
130
what was used to determine that oxamate triggers loop closure and the rate of closure
pre steady state kinetics of the E-NADH complex with oxamate
131
what was the rate of loop closure and what did this show
125/s
| slow step in the reaction
132
what caused the apparent substrate protection upon the addition of NEM, now suggesting cya 165 isn't in fact involved in catalysis
loop closure prevented cys165 from being accessible to NEM
133
what is the michaelis menten equation with relation to Kcat
V=Kcat[E][S]/km[S]
134
what is the equation for the specificity constant
kcat/km
135
how do you find the relative rate of an enzyme with two diff substrates
ratio of kcat/km
136
what did scientists try to change the specificty off ldh to
oxaloacetate and malate
137
what did they not want to change in the experiment to change the specificity of ldh
anything to do wit nad/h binding
binding of carboxalate groups
mechanism
loop closure
138
what is the diff between pyruvate and oxaloacetate
bigger and more negative
139
what were the focus features of the experiment
charge balance
substrate size vs active site volume
direct electrostatic complementarity
140
what was the overall charge in the wt reaction compared to mutant and what did this mean for the experiment
0 and -1
| need to change active site to be less negative
141
what 2 mutations were introduce to make active site less negative and what were the results
D197N- less with P but same with O
| E107Q- lees activity with P, more with O but negligible
142
was E or D a buried residue
D- larger effect with exposed residue
143
how was the pocket made larger and what was result
thr246gly
| greatly decresed w P, slightly increased with O
144
which mutation produced a huge switch in specificity
Gln102Arg
145
what is glutathione made up of
g-glutamate, cysteine and glycine
146
what is the oxidised version of glutathione called
GSSG
147
what reaction does glutathione reductase catalyse
GSSG + NADPH + H --> 2GSH + NADP
148
which family of proteins does glutathione reductase belong to
flavoprotein disulphide oxidoreductases
149
how many subunits aew in glutathione reductase
2 (dimeric)
150
what was the aim of the protein engineering experiments with glutathione reductase
change specificity from nadph to nadh
151
what is the active site of glutathione reductase divided by
fad
152
what do we not want to change in glutathione reductase
anything to do with glutathione binding
| anything involved in binding fad
153
what do we want to change in glutathione reductase
how it binds nadph0
nad smaller and less negative
focus on very positive bnding pocket
154
how many mutations were required to change specificity
7
