Enzyme mechanisms

B. X-ray methods
Small angle X-ray scattering (SAX)
Kinetic crystallography – slow reactions
Kinetic crystallography – Laue method
Kinetic crystallography – freezing techniques
Anomalous dispersion

1
 Enzyme mechanisms
B. X-ray methods
Small angle X-ray scattering (SAX):
Waves are scattered at objects with which they interfere.
The finer the lattice (i.e. small objects), the larger the
scattering angle. X-rays (electromagnetic waves) are
scattered at objects of molecular size (nm) with varying
electron density.

X-ray direct beam

detected signal
2
 Enzyme mechanisms
B. X-ray methods
Small angle scattering: MurA
(UDP-N-acetylglucosamine enolpyruvyltransferase)
The enzyme has two domains, which are in a closed
conformation (with substrate or inhibitor) or an open
conformation (without ligand) – in the crystal structures.

Are the differences an artifact of the crystal structure or of

relevance for the solution structure?

3
 Enzyme mechanisms
B. X-ray methods
Small angle scattering: MurA
open modelled closed closed
(determined later)

4
 Enzyme mechanisms
B. X-ray methods
Small angle scattering: MurA
Complex with pyruvate-P (•)

(1) Fitted with open structure

(2) Fitted with closed model
(3) Fitted with closed structure

5
 Enzyme mechanisms
B. X-ray methods
Small angle scattering: MurA

-s
Protein solution without and

ub
st
fit open
with UDP-glucosamine

ra
te
+s
ub
st
Fitted with open structure

ra
te
Fitted with closed structure

fit closed

6
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography – slow reactions

Remember: L-Haloacid dehalogenase. Trapping of the

covalent intermediate by cryocooling (‚freezing‘) during
the reaction.
Data collection needs to be faster than preparation /
soaking / reaction.
Data collection: < 10 min at a strong X-ray source for
data set, s for single frames, ns - ms for Laue frames.
Preparation: s, Soaking: 10-100 s, Reaction: ?

7
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography – Laue method

Normally only single wavelength X-ray light is used for

diffraction experiments: ca. 100 frames / data set.
With white X-rays (l = 0.8-2 A) only few frames are
needed and the intensity is higher: ms / frame.

8
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography – Laue method
Photoactive yellow protein: Light triggers a
conformational change in the protein.
After a short laser puls (ns), a Laue photograph (ns) is
taken. The protein relaxes and the procedure is
repeated.

9
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography –
Laue method

Photoactive yellow protein:

The photocycle

10
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography –
Laue method

Photoactive yellow protein:

pG groundstate
chromophore H-bonded to
Tyr42
trans double bond,

11
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography –
Laue method

Photoactive yellow protein:

pR = first excited state
at 1- 1.2 ns after
excitation
chromophore H-bonded to
Tyr42
cis double bond,

12
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography – Laue method
Photoactive yellow protein: pB = second excited state at
2-12 ms after excitation
chromophore H-bonded to Arg52, cis double bond

13
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography – Laue method
Photoactive yellow protein: The photocycle

pG pR pB 14
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography – freezing techniques
A crystal of a protein-substrate complex is cryocooled
(or a photoactivatable precursor: caged compound).
The reaction is started, e.g. by laser.
The reaction cannot proceed, because motions are frozen.
At increasing temperature further steps may be
enabled.

15
 Enzyme mechanisms
B. X-ray methods
Kinetic crystallography – freezing techniques
Myoglogin: A crystal with a CO complexed to heme is
irradiated with a laser. CO dissociates from the heme.
But: due to the low temperature, the CO cannot diffuse
out of the binding pocket. At higher temperature the CO
can be seen on its way out.

16
 Enzyme mechanisms
B. X-ray methods
Anomalous dispersion:
Normally the incident X-ray continues without phase shift
after the scatterer.
If the energy is at or above the absorption edge (energy to
eject an electron from the atom) of the scatterer, a
phase shift occurs: anomalous scattering.

The anomalous
scattering is quantified
by f‘‘. It is strongly
wavelength dependent.
17
 Enzyme mechanisms
B. X-ray methods
Anomalous dispersion:

Zn
Cu

E
Use this energy to see an effect from
Cu and Zn

Use this energy to see an effect only

from Cu
Only normal scattering 18
 Enzyme mechanisms
B. X-ray methods
Anomalous dispersion:

Zn

Zn
Cu
Cu

Electron density calculated
from normal scattering.
‚Anomalous electron
density‘ calculated from
anomalous scattering at
19
‚Cu and Zn‘ wavelength.