NMR Relaxation Dispersion Studies of Large Enzymes in Solution - Recent Developments in Biomolecular NMR - page 161

13 C MQ-CPMG relaxation dispersion for I83d in IGPS bound to
PRFAR. The experiment was acquired at 14.1 T and 303 K. Dispersion
data was fit with eqn (7.11) and gives k ex 5 500 ¡ 220 s 21 .

Figure 7.4

dispersion data with eqn (7.11) gives a k ex 5 500 ¡ 220 s 21 , which is similar to
that for the amide backbone (Figure 7.3). The similarity in exchange rate
constants between side-chain and backbone positions further suggests a
concerted motion for these residues and also provides a more complete picture
of ms motions than the individual experiments.

7.5.2 Triosephosphate Isomerase
Triosephosphate isomerase (TIM) is a 53 kDa homodimeric enzyme of the
glycolytic pathway. TIM catalyzes the reversible isomerisation of glyceralde-
hyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). TIM has
served as a model for understanding the role of conformational changes in
enzyme function. TIM possesses a highly conserved 11 amino acid active-site
loop (loop 6) that must remain open to bind substrate and release product but
that also must close and sequester the active site during the chemical
reaction. 68-72 This structural information coupled with biochemical study of
the catalytic rate for this reaction places a lower limit on the timescale for this
loop movement; the loop cannot move slower than the overall catalytic
turnover rate. This timescale, unfortunately places loop 6 motion outside the
regime to which the CPMG dispersion experiment is sensitive. Its motion was
therefore characterised with the TROSY-selected off-resonance R 1r experi-
ment (Figure 7.5). 7,73 Fits of the R 1r dispersion data for the N-terminal loop 6
residue V167, with the fast-limit form of eqn (7.8), provided a k ex value equal
to 8900 ¡ 1600 s 21 , a value that is nearly identical to k cat for this enzyme. This