Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Isomerases 229
Recent studies on mandelate racemase have provided an insight into this
problem. Mandelate racemase is a cofactor-independent enzyme that catalyses
the interconversion of R- and S-mandelic acid. A two-base mechanism has been
implicated for this enzyme, and is supported by an X-ray crystal structure of the
enzyme in which active site residues Lys-166 and His-297 are suitably positioned
to act as the two bases (see Figure 10.4). Replacement of His-297 by
glutamine using site-directed mutagenesis gives a mutant enzyme that is unable
to catalyse the racemisation reaction. This mutant enzyme (still containing
Lys-166) is able to catalyse the exchange of the a-proton of S-mandelic acid
with 2 H 2 O to give 2 H-S-mandelic acid, but does not catalyse exchange with
R-mandelic acid. This result implies that Lys-166 deprotonates the a-proton of
S-mandelic acid, forming a carbanion intermediate. This intermediate is suYciently
stable to exchange with the e-NHD þ 2
of Lys-166.
How is the carbanion intermediate stabilised It was evident from the X-ray
crystal structure that the carboxylate group of the substrate forms hydrogen
bonds with a protonated Lys-164 residue and a protonated Glu-317 residue.
Replacement of Glu-317 by a glutamine residue using site-directed mutagenesis
gives a mutant enzyme with 10 4 -fold reduced catalytic eYciency. It has been
Figure 10.4 Structure of mandelate racemase (PDB Wle 2MNR), showing the a,b-barrel structure of
the protein. Catalytic bases Lys-166 and His-297, and active site residues Lys-164 and Glu-317, are
shown in red. Bound SO 2 4 ion shown in black.