Reaction Mechanism and Catalytic Fingerprint of Allantoin Racemase

The
stereospecific oxidative decomposition of urate into allantoin
is the core of purine catabolism in many organisms. The spontaneous
decomposition of upstream intermediates and the nonenzymatic racemization
of allantoin lead to an accumulation of (R)-allantoin,
because the enzymes converting allantoin into allantoate are specific
for the (S) isomer. The enzyme allantoin racemase
catalyzes the reversible conversion between the two allantoin enantiomers,
thus ensuring the overall efficiency of the catabolic pathway and
preventing allantoin accumulation. On the basis of recent crystallographic
and biochemical evidence, allantoin racemase has been assigned to
the family of cofactor-independent racemases, together with other
amino acid racemases. A detailed computational investigation of allantoin
racemase has been carried out to complement the available experimental
data and to provide atomistic insight into the enzymatic action. Allantoin,
the natural substrate of the enzyme, has been investigated at the
quantum mechanical level, in order to rationalize its conformational
and tautomeric equilibria, playing a key role in protein–ligand
recognition and in the following catalytic steps. The reaction mechanism
of the enzyme has been elucidated through quantum mechanics/molecular
mechanics (QM/MM) calculations. The potential energy surface investigation,
carried out at the QM/MM level, revealed a stepwise reaction mechanism.
A pair of cysteine residues promotes the stereoinversion of a carbon
atom of the ligand without the assistance of cofactors. Electrostatic
fingerprint calculations are used to discuss the role of the active
site residues in lowering the pKa of the
substrate. The planar unprotonated intermediate is compared with the
enolic allantoin tautomer observed in the active site of the crystallized
enzyme. Finally, the enzymatic catalysis featured by allantoin racemase
(AllR) is compared with that of other enzymes belonging to the same
family.