Biomedical Engineering Reference
In-Depth Information
separated by 30
˚
in which the ammonia (NH
3
) product from the first
reaction, generated by hydrolysis of glutamine, is required in the second
reaction, which involves coupling of the NH
3
with N9-[(59-phosphoribulosyl)-
formimino]-5-aminoimidazole-4-carboxamide-ribotide (PRFAR) to generate
imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide
ribotide (AICAR).
65,66
There is a 10
3
-fold increase in the rate of Gln hydrolysis
when PRFAR is bound at the distant active site.
67
This is an example of V-type
allostery and the mechanism of communication between these two active sites
was characterised by solution NMR dispersion experiments.
14
7.5.1.1
15
N SQ-CPMG Dispersion
Shown in Figure 7.3 are two residues, E87 and R16, located near the PRFAR
binding site that exhibit ms motions when PRFAR is bound but have flat
dispersion curves in its absence. Fits of the CPMG relaxation data with eqn
(7.7) give similar exchange rate constants suggesting the presence of concerted
motion; motion that is activated by the binding of the allosteric effector and
substrate PRFAR.
7.5.1.2
13
C MQ-CPMG Dispersion
In a similar fashion, ILV MQ
13
C dispersion data indicate an enhancement of
methyl side-chain motions when PRFAR is bound.
14
Figure 7.4 shows a
13
C-
MQ dispersion curve for I83d in the presence of PRFAR. Fits of the CPMG
Figure 7.3
15
N TROSY-CPMG relaxation dispersion curves for E87 and R16,
indicated as black spheres, in IGP synthase bound to substrate PRFAR
(shown in stick representation). Individual k
ex
values from fits to these
dispersion data were 380 ¡ 80 s
21
and 490 ¡ 80 s
21
for E87 and R16
respectively. The similarity in k
ex
values between these and other (not
shown) residues suggests concerted motion for select residues in this 52
kDa enzyme. Data were acquired at 14.1 T and 303 K.
