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2. BIOC 2306 S1 > Enzymology > Flashcards

Enzymology Flashcards

1
Q

What is the transition state theory?

A

The transition state is the highest energy intermediate
= the least populated state
For catalysis to occur, the enzyme must bind the transition state more tightly than it binds the substrate
The more tightly an enzyme binds the TS relative to the substrate, the greater the rate

Transition state barrier - this determines what the rate of reaction will be

TS theory proposes - the rate determining step in the reaction is the rate of decomposition of the transition state

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2
Q

What can kinetic equation can we derive for the transition state theory?

A

The transition state (X‡) is in rapid equilibrium with the reactants
A + B ⇌ X‡ → P + Q
We can get the equilibrium constant:
K‡ = [X‡] / [A][B]

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3
Q

Describe the effects of enzymes preferentially binding to the transition state?

A

An enzyme may bind the transition state of the reaction it catalyzes with greater affinity than its substrates or products

Enzymes that preferentially bind the transition state structure increase its concentration and therefore proportionally increase the reaction rate

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4
Q

How can the transition state be achieved?

A

If there is stress/stain on surrounding groups it can push other groups together, distorting the bond angles making the molecule look like the transition state

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5
Q

What is the effect of a catalyst?

A

The energy barrier is lowered = lowers the height of the transition state

Energy difference between the catalysed and uncatalysed - this change greatly increases rate enhancement

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6
Q

What is some proof of the transition state theory?

A

Many drug designs are based on transition state theory
Enzymes have evolved to bind TS more tightly than substrate
Therefore competitive enzyme inhibitors must resemble the TS, rather than the substrate

They are called transition state analogues

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7
Q

What are some examples of transition state analogues?

A

Abzymes - catalytic antibodies
Make an analogue of the transition state, inject into an animal and they can produce antibodies against the TS analogue
The antibodies are purified - they have a empty binding site that can then catalyse a reaction by binding to the transition state

Molecular imprinting
Polymers are molecularly imprinted with transition state analogue template so the gel can function as catalysts
Doesn’t work very well

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8
Q

What are multiple substrate reactions?

A 
Bisubstrate reactions are reactions with multiple substrates yielding multiple products
Ordered sequential Bi Bi
Random Ordered Bi Bi
Bi Bi Ping Pong
Theorell Chance
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9
Q

Describe the Bisubstrate reaction types?

A

Involving a ternary complex, occur via single displacements:
Ordered formation - compulsory order of the substrate addition to the enzyme
Random - both binding sites are present on the free enzyme (any order)

Not involving a ternary complex, occur via double displacements:
Ping Pong - group-transfer reactions in which one or more products are released before all substrates have been added (substrates don’t encounter each other on the surface of the enzyme)
Theorell-Chance - intermediate doesn’t really exist it displaces that quickly

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10
Q

What are some examples of the bisubstrate types?

A

Ordered sequential Bi Bi - NAD(P)(H)-linked dehydrogenases
Random Ordered Bi Bi - No common examples
Bi Bi Ping Pong - Pyridoxal phosphate dependent transaminases
Theorell Chance - Horse liver alcohol dehydrogenase

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11
Q

What are the kinetic equations?

A

v = k[EQ]

We don’t need to learn the kinetic equations - written on cards

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12
Q

What is the significance of the parameters within the kinetic equations of bisubstrate reactions?

A

v, [A] and [B] are variables

Vmax, KA, KB and K’A are parameters

Vmax, the maximal rate of the reaction
Achieved when both [A] AND [B] are saturating

KA and KB - The Michaelis constants Km for A or B, respectively

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13
Q

How do we gather two substrate kinetic data?

A

We use very similar methods/assays such as spectrophotometry
Plot - Time v observed activity - to find the initial rate

Keep A concentration the same but change B - measure the rate of each reaction, around 5 times
Then keep B the same but change A
Do this multiple times

End up with a table 5x5

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14
Q

How do we process two substrate kinetic data?

A

We take the inverse of the kinetic equation and then cancel and rearrange it into a y=mx+c format
We plot these figures to form a primary plot 1/v against 1/[A]
We can measure the slopes and intercept on the primary plot

Now take the m part of the equation,rearrange that in to y=mx+c and plot that
Then on another graph take the c part of the equation, rearrange that into y=mx+c and plot that

From there you can work out the Vmax and Km - look at written flashcards

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15
Q

What do the primary plots look like for the different types of bisubstrate reactions?

A

Ordered Sequential Bi Bi reaction - different slopes and different intercepts
They cross after the Y axis but before the X axis (looks like a fan)

Ping Pong Bi Bi reaction - parallel slopes with different Y intercepts

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16
Q

How can we determine kinetic mechanisms?

A

Find partial reactions - if a reaction is in two parts
Isolation of modified enzyme
Find product inhibition patterns

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17
Q

How can we determine a partial reaction e.g. which is substrate [A]?

A

Equilibrium dialysis (you could use surface resonance dialysis)

Take a semi permeable bag filled with enzyme into a large tank of buffer and substrate
The enzyme it is too big to move out of the bag
Substrate will diffuse in and out - until equilibrium
Some part of Enzyme will become ES complex
Therefore [S]in > [S]out - as there is free substrate as well as ES substrate in the bag

Example: Lactate dehydrogenase - binds NAD+ and no visible binding of lactate - showing it is an ordered reaction and NAD+ = A

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18
Q

Why can we use product inhibition patterns to help determine the kinetic method?

A

Since all reactions are reversible to some extent, and since the products are formed in the reaction by the enzyme, it follows that the products of the reaction (P and Q) must bind to the enzyme and can therefore act as inhibitors

Very [A] or [B] keeping the other constant and look at the effects of either [P] or [Q]
As the substrates can be saturating or non-saturating values
There are 8 possible combinations

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19
Q

What are the rules for product inhibition patterns?

A

Rule 1
The ordinate intercept (the y-axis intercept) of a reciprocal plot is affected by a compound which associates reversibly with an enzyme form other than the one with which the variable substrate combines

Rule 2
The slope of a reciprocal 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

Note:
Reversible steps are considered to be all steps except when:
Saturation with substrate blocks the reversible step
When the other product is involved and we consider that its concentration = 0

20
Q

What is significant about product inhibition patterns?

A

These are more effective in determining the kinetic mechanism but each square needs at least 25 points as you need to vary [A] and P for example around 5 times for plots

21
Q

How can we determine which amino acid residues are specifically involved int stabilisation of the TS within the active site?

A

We have to do multiple experiments to build up a full picture of how an enzyme functions

We need to look at/do:

  1. Protein chemistry
  2. Kinetic studies
  3. Chemical modification of amino acids
  4. Stereochemistry
  5. X-ray crystallography
  6. Site-directed mutagenesis
22
Q

What should we look for within protein chemistry?

A

E.g. LDH

Position of enzyme - cytosolic
Mr - 140,000
Type - tetrameric
Isoenzyme? - yes
We can then study the different forms depending on where in the organism they reside

e.g. We can use this to determine the difference between a heart attack or ruptured intercostal muscles
As after a heart attack the LDH isoform can leak into the plasma and be identified

23
Q

What kinetic studies can we do?

A

We can determine type of mechanism reaction e.g. Ordered Sequential Bi-Bi

And then produce a Cleland Diagram
So we can invisage each individual rate constant between each intermediate - look at pre-steady state kinetics

24
Q

What are pre-steady state kinetics?

A

Concerned with the formation and consumption of ES intermediates within the first few milliseconds of the reaction until their steady state concentrations are reached

25
Q

How can we measure pre-steady state kinetics?

A

Continuous-flow spectrophotometer:
In two separate syringes - enzyme and substrate
As they meet they mix, and as we move further down the tube the has been more reaction that has taken place
Therefore if we measure earlier down the tube - the distance is short - less time to react and we can measure the reaction using light

This is expensive as it has to be continuously flowing

26
Q

How do we now measure pre-steady state kinetics?

A

Stopped-flow spectrophotometer
We simultaneously push the reactants/enzyme in and it hits the ‘wall’ and stops
So the reaction happens and we can measure that specific point - not against time
The driving force is a nitrogen cylinder - in order to throw the liquids together and measure these rapid reactions

27
Q

Describe the data collected from pre-steady state kinetics?

A

There is a burst phase - ‘dead time’ of the instrument
And then we go back to the steady state

The ‘burst’ is the entire forward reaction happening immediately
The remaining slow increase is the next NADH being formed, then the next, then the next
It can only move as fast as the slowest step
Determining the rate determining state is very hard to nail down

We can do multiple experiments with different intermediate elements as the ‘reactants’ in the stopped-flow spectrophotometer

28
Q

Describe types of chemical modifications of amino acids?

A

To find which residues are in the active site and what is their function

Affinity labels
Modify the enzyme in the active site due to special affinity of the compound to the particular enzyme

General labels
Modify specific types of amino acid
Histidine labels
Arginine labels
Cysteine labels
29
Q

Describe a type of affinity labels?

A

A substrate analogue e.g. Bromopyruvate

This should bind but the Br may covalently bind to an essential residue which would then affect the function of the enzyme

So…
Unfold the protein
Digest it then separate and sequence
We can identify the residue that binded to the Br (affinity tag)
Bromopyruvate - His195 is modified - therefore has something to do with the catalysis

30
Q

What are some general labels?

A

Diethylpyrocarbonate (DEPC)
It inactivates the enzyme upon covalent modification
Isolate modified peptide - sequence and identify this modified residue = His195

Phenylglyoxal
Treat LDH with this - it inactivates it
This shows that a phenylalanine is somehow responsible in catalysis of this enzyme
The modified peptide isn’t stable enough for isolation - therefore don’t know the exact residue

N-ethyl maleimide (NEM)
Modifies cysteine residues
Inactivated - Cys-165 (stable enough)
Involved in substrate-protection

31
Q

What is substrate-protection?

A

In the presence of the substrates the enzyme is protected as the label doesn’t have room to get into the active site

32
Q

Describe stereochemistry?

A

The arrangement of atoms in space, to show their 3D structure

This can tell us the relative chemical orientation of the two substrates

33
Q

Why is stereochemistry important?

A

Example: Alanine
In the L-form all 3 groups can bind amongst itself within the active site
In the D-form all 3 groups can’t bind, no matter the orientation within that form

34
Q

What are the types of stereochemistry?

A

Chiral - all 4 groups around the C atom are different

Pro-chiral - can be converted to chiral in one step (therefore have 2 of the same group e.g. H)

35
Q

Describe the stereochemistry of LDH?

A

It is Pro-Chiral with 2 H groups
By disallowing rotation of N-R we can distinguish between the 2 H’s
We can prevent N-R rotating by having a binding site around the right large side chain

‘A-side specific’ – HA removed
e.g. Malate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase

‘B-side specific’ – HB removed
e.g. Glutamate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase

36
Q

Describe X-ray crystallography?

A

A narrow parallel beam of x-rays are directed at a well ordered protein crystal
The atoms in the crystal scatter some of the beam and the scattered waves reinforce one another at certain points - appearing as a pattern of diffraction spots by a detector

The diffraction pattern as well as the amino acid sequence can be used to produce an atomic model
A simplified version of the atomic model shows the proteins structual features clearly

37
Q

Describe the X-ray crystallography of LDH?

A

By seeing all the atoms in the enzyme and the substrates - we can see which residues are involved
This enzyme has two domains within each of the 4 monomers

N-terminal domain - binds NAD+
This N-terminal domain is found in many dehydrogenases (Rossmann fold or the dinucleotide binding domain)
6-stranded parallel bsheet + 4 a-helices

C-terminal domain - binds pyruvate/lactate
Two 3-stranded antiparallel sheets

Most of the dehydrogenases contain a Rossman fold - but it doesn’t have to lie within the same places
It was recruited during evolution - but incorporated in different ways (divergent evolution)

38
Q

How do we see the interactions between the enzyme and substrates in X-ray crystallography?

A

We need to see the interaction with the substrates bound
This is difficult as we attached they should be catalysed to form into products
We use binding inhibitors/substrate analogues to study these interactions e.g. Oxalate and Oxamate
These mimic the position of the binding of the substrates but they won’t turnover

39
Q

What is the x-ray crystal interactions of the NADH structure?

A

Structure of NADH - Adenine + ribose = Adenosine
Adenosine + pyrophosphate + ribose + nicotinamide ring

There is a strong interaction with Lysine250 with the =O in the nicotinamide ring
This prevents the rotation of the N-R at the bottom of this ring - allowing stereochemical determination

OH at the bottom left of the ribose in the adenosine - binds Glu-140 via H bonding (makes LDH specific for NADH and not NADPH)
As a phosphate would repel the Glu-140 and not make an interaction

40
Q

What interactions does pyruvate contain with LDH?

A

Arg171 and Arg109
His195
It is a surprise that 2 Arg are used as our labelling only showed 1 was used but not even which one - therefore X-ray crystallography is essential

41
Q

What is X-ray crystallography essential for?

A

Crystallography shows that conformational change is critical to catalysis
Especially going from E-NAD to E-NAD-Lactate
During this process Arg-109 moves around 1.4 nm and makes better contact with the substrate

42
Q

What is Site-directed mutagenesis used for?

A

We can explore and test our mechanistic hypotheses using site directed mutagenesis

Example: Arg-109 -> Gln-109 (R109Q)
After substitution, do a 25 point matrix, plotting primary/secondary plots to get to the Km and Kcat
R109Q – no change in NAD binding
R109Q – catalysis rate falls x400
R109 – important in catalysis (it helps stabilise the transition state)

43
Q

How can we view site-directed mutagenesis and loop closure?

A

Tryptophan is the most fluorescent amino acid
Useful way of monitoring the environment around the tryptophan

LDH contains 3 Trp residues and we are going to mutate them
W80Y, W150Y, W203Y variant is non-fluorescent but active
Place a new Trp in loop - G106W
We can see if the loop will be closed or open

To test we look at the pre-steady state kinetics - measuring the fluorescence
We can then work out the rate constant for the fluorescence change

We found the slow step is the conformational change overall

44
Q

Describe the overall mechanism of LDH?

A 
LDH binds NADH
Many interactions eg Lys250
LDH-NADH binds lactate – Arg171 important
Loop closure
Brings Arg109 into active site
Helps transition state stabilization
His195 in close proximity
H transferred from NADH
His195 protonates
NADH/Pyruvate formed
Pyruvate released
NADH released
45
Q

What wasn’t included in the end mechanism of LDH?

A

The role of Cys165 - as it was inactivated by NEM
Cys165 is not involved in mechanism/catalysis
It doesn’t take a part in catalysis - but it is close enough on the edge of the active site to interfere

An important lesson: we should always gain multiple bits of evidence to support our hypotheses
Each result supports our hypothesis and contrary results suggest we should modify our theory

2. BIOC 2306 S1 (19 decks)

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