During the course of our study of general acid/base catalysis,
Asn485 was mutated to leucine in order to determine whether it
was contributing to either the oxidation or isomerization reactions.
Our kinetic results indicated that Asn485 plays a crucial role
in the oxidation activity of the enzyme. When Asn485 was substituted
with a leucine, the oxidation activity was greatly impaired. Steady-state
kinetic analysis showed that kcat for oxidation is 1000-fold slower
in N485L than in wild type, but is only 20-fold slower for isomerization.
We examined the X-ray crystal structure of the N485L mutant in
order to determine the effect of the mutation on the active site
structure. We observed a small shift in the position of the water
structure in the active site. By analogy, the substrate position
would be similarly shifted and the substrate may no longer be
ideally aligned with Glu361 and the FAD. However, the markedly
lower oxidation activity of N485L relative to isomerization implied
that the major effect of the mutation is not primarily due to
the misalignment of the substrate in the active site.
Although the N485L reaction is three orders of magnitude slower
than wild type, primary deuterium kinetic isotope effects showed
that 3a-H transfer is still rate-determining
in the mutant reaction. This means that mutation of Asn485 reduces
the rate of 3b-hydroxy oxidation, rather
than decreasing the rate of another kinetic step, and that Asn485
is important for FAD reduction. In contrast, when His447 is mutated,
3a-H transfer is no longer rate determining (12).
The structural and kinetic data for N485L suggested that Asn485
is important for creating an electrostatic potential around the
FAD that is favorable for oxidation of alcohol substrates. Indeed,
the UV/vis spectrum of the FAD region of N485L is red-shifted
relative to wild-type, indicating that the electronic environment
around the FAD has been altered. In addition, the mutation of
Asn485 to leucine and its associated structural changes resulted
in a 76 mV decrease in the mid-point reduction potential of the
FAD. In other words, the N485L mutant is a much poorer oxidizing
agent, and the reduction of the N485L-bound FAD is not as thermodynamically
favorable as that of wild-type. This is consistent with the kinetic
properties that we observed, i.e., a higher activation barrier
to FAD reduction.
Figure 3: Molscript (2) view
of helix-14. The bonds for the pyrimidine ring of the FAD cofactor
are colored yellow, those for the side chains of Asn485, Pro486
and Met122 are in black and the secondary structure elements are
shown in gray. The side chains and the pyrimidine ring are located
at the N-terminus of the helix.
The change in reduction potential observed corresponds to approximately
3 kcal/mol transition state stabilization energy after correcting
for other changes that occur in the active site. The interaction
is an amide-p interaction between the
asparagine side-chain and the p-system of the pyrimidine of the
FAD. Furthermore, the helix 14 dipole amplifies the strength of
this interaction (Figure 3). Empirical potential energy calculations
have suggested that an N-H···p
interaction has a stabilization energy of approximately 3 kcal/mol
(22). QM/MM calculations on the cholesterol oxidase system estimate
that 2 kcal/mol stabilization energy may be derived from the interaction
(23). This magnitude range is qualitatively consistent with the
rate reduction that we observed. Although many protein-flavin
interactions have been studied in different systems, this is the
first example, to our knowledge of a p-cation-like interaction
between the isoalloxazine ring and the protein.
