Modification of intersubunit interactions in DAAO
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
275
Table 3. Effective rate constants for inactivation of R220E
TvDAAO and R169A/R220A TvDAAO at different temperatures
the removal of hydrogen bonds involved in the intersubꢀ
unit contact leads to the transformation of TvDAAO in
the completely insoluble form. We plan to perform amino
acid substitutions that would enhance the intersubunit conꢀ
tact in the TvDAAO molecule and lead to an increase in
thermal stability of the enzyme.
Mutant enzyme
TvDAAO R220E
Т/ °С
k1
k2
min–1
32 0.184 0.023 0.0078 0.0019
34 0.253 0.08
36 0.490 0.11
38 0.730 0.18
40 1.000 0.26
0.020 0.005
0.039 0.008
0.047 0.013
0.068 0.015
The present study was supported by the Russian Founꢀ
dation for Basic Research (Project No. 08ꢀ04ꢀ01703) and
the Federal Agency for Science and Innovations of the
Russian Federation (Contract 02.512.11.2299).
TvDAAO R169A/R220A 20 0.250 0.004 0.0096 0.0024
22 0.480 0.10
24 0.510 0.12
26 0.770 0.15
28 0.960 0.17
0.032 0.005
0.044 0.008
0.061 0.110
0.076 0.016
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evident from the aboveꢀconsidered data, the effective rate
constant k1 characterizing the dissociation of the dimer
more slowly increases with temperature than the effective
rate constant k2 characterizing the rate of inactivation of
the monomer, i.e., the energy of activation for the second
step is higher than that for the first step. This is in good
agreement with our model of inactivation of TvDAAO,
because the energy of activation for the dissociation of the
dimer into individual subunits should be lower than that
necessary for the unfolding of the protein globule in the
step of inactivation of the monomer.
The aboveꢀconsidered data indicate that the arginine
residues in positions 169 and 220 play a considerable role
in the formation of the quaternary structure and ensuring
the stability of TvDAAO. The substitution of other amino
acid residues for these residues results in that the enzyme
is synthesized in the active but insoluble form. The introꢀ
duction of the Arg220Glu mutation into the TvDAAO
molecule substantially impairs the kinetic parameters with
different substrates and leads to a decrease in stability
of the enzyme. In the case of the double substitution
Arg169Ala/Arg220Ala, the higher constants KM with reꢀ
spect to a series of Dꢀamino acids were observed in spite of
the fact that the kinetic properties were studied for the cell
wallꢀbound enzyme. It should be noted that substantial
changes in the substrate specificity profile were caused by
the substitutions of the amino acid residues that are not
involved in the active site of the enzyme. An analoꢀ
gous effect was observed in the case of the Cys108Phe,
Cys108Ala, and Cys108Ser single mutations.13 The first
mutation leads to an increase in thermal stability of
TvDAAO, whereas the second and third mutations cause
a decrease in thermal stability.
The results of our experiments also indicate that the
existence of TvDAAO in the dimeric form is more critical
for its stability compared to the enzyme from the yeast
R.gracilis. In the latter case, the monomeric mutant enꢀ
zyme was obtained in the active and soluble form, whereas
Received August 26, 2009