Page 19 - ILLS Annual Report 2018

SCIENTIFIC HIGHLIGHTS
16-17
Figure 2
Conventional mechanism of aspartate aminotransferase [3].
The neutron structures revealed that protonation states of the phenolic oxygen and the Schiff bases are incorrect in the internal and external aldimine states.
was now protonated, while keeping the protonation
states of the pyridine nitrogen and the phenolic oxygen unchanged. This could only happen if the substrate came in as a zwitter-ion, with the N-terminal nitrogen protonated and the C-terminal carboxylic group deprotonated. Quite unexpectedly, the Schiff base hydrogen of the external aldimine was observed located halfway, and thus shared, between the nitrogen and an oxygen of the carboxylate in an apparent low-barrier hydrogen bond.
Quantum chemistry calculations [1, 2] using the neutron structures to construct accurate theoretical models have
demonstrated no involvement of the ground state strain or destabilisation to explain the power of aspartate aminotransferase, overturning previous mechanistic proposals. Instead, favourable stereo-electronic effects
in the internal and external aldimine states contribute significantly to the enzyme’s ability to lower the transition state energy, increasing the rate of catalysis by as much as 1012 times.
These results demonstrate that neutrons are indispensable for biochemical investigations to identify the molecular determinants of the catalytic power of enzymes.
Figure 3
The neutron structures of aspartate aminotransferase in the internal (left) and external (right) aldimine states. The nuclear density map for the PLP co-factor and surrounding residues is shown as a grey mesh. The omit nuclear density map (magenta mesh) shows exact locations of hydrogen atoms observed as deuteriums.
Figure 1
Chemical structures of pyridoxal 5’-phosphate (PLP) co-factor and its covalent adducts with the catalytic lysine (Lys) residue and a substrate (R is -CH2COO− for L-aspartic acid).
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