Myrosinase from
Sinapis alba
hydrolyzes glycosidic
bonds of β-
d
-
S
-glucosides. The enzyme
shows an enhanced activity in the presence of
l
-ascorbic
acid. In this work, we employed combined quantum mechanical and molecular
mechanical (QM/MM) calculations and molecular dynamics simulations
to study the catalytic reaction of wild-type myrosinase and its E464A,
Q187A, and Q187E mutants. Test calculations show that a proper QM
region to study the myrosinase reaction must contain the whole substrate,
models of Gln-187, Glu-409, Gln-39, His-141, Asn-186, Tyr-330, Glu-464,
Arg-259, and a water molecule. Furthermore, to make the deglycosylation
step possible, Arg-259 must be charged, Glu-464 must be protonated
on OE2, and His-141 must be protonated on the NE2 atom. The results
indicate that assigning proper protonation states of the residues
is more important than the size of the model QM system. Our model
reproduces the anomeric retaining characteristic of myrosinase and
also reproduces the experimental fact that ascorbate increases the
rate of the reaction. A water molecule in the active site, positioned
by Gln-187, helps the aglycon moiety of the substrate to stabilize
the buildup of negative charge during the glycosylation reaction and
this in turn makes the moiety a better leaving group. The water molecule
also lowers the glycosylation barrier by ∼9 kcal/mol. The results
indicate that the Q187E and E464A mutants but not the Q187A mutant
can perform the glycosylation step. However, the energy profiles for
the deglycosylation step of the mutants are not similar to that of
the wild-type enzyme. The Glu-464 residue lowers the barriers of the
glycosylation and deglycosylation steps. The ascorbate ion can act
as a general base in the reaction of the wild-type enzyme only if
the Glu-464 and His-141 residues are properly protonated.