J. Am. Chem. Soc. 2001, 123, 11325-11326
Contribution of Phosphate Intrinsic Binding Energy
11325
to the Enzymatic Rate Acceleration for
Triosephosphate Isomerase
Tina L. Amyes, AnnMarie C. O’Donoghue, and
John P. Richard*
Department of Chemistry, UniVersity at Buffalo
SUNY, Buffalo, New York 14260-3000
ReceiVed August 2, 2001
We report that 80% of the enzymatic rate acceleration for the
prototypical proton transfer from carbon catalyzed by triosephos-
phate isomerase can be directly attributed to the remote phos-
phodianion group of the substrate (R)-glyceraldehyde 3-phosphate,
and that the intrinsic binding energy of this functional group in
the transition state for enzyme-catalyzed enolization is 14 kcal/
mol.
Figure 1. Logarithmic timecourses for the disappearance of 11 mM (R)-
glyceraldehyde catalyzed by 0.17 mM rabbit muscle TIM at pD 7.0 in
D2O at 25 °C and I ) 0.1 (NaCl). Values of kobsd (s-1) were obtained
from the least-squares slopes (solid lines). Key: (b) In the presence of
24 mM imidazole buffer. (9) In the presence of 18 mM imidazole buffer
and 8 mM 2-phosphoglycolate (Ki ≈ 5 µM).12,13
Triosephosphate isomerase (TIM) catalyzes the reversible
stereospecific aldose-ketose isomerization of (R)-glyceraldehyde
3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) by
a single-base (Glu-165) proton-transfer mechanism through a cis-
enediol(ate) intermediate (Scheme 1).1-5 TIM is a paradigm for
enzymatic catalysis of proton transfer at carbon, and catalyzes
isomerization of the free carbonyl form of GAP to DHAP at the
Scheme 1
diffusion-controlled limit with kcat/Km ) 2.4 × 108 M-1 s-1 2,3
.
We consider here the possibility that the large enzymatic rate
acceleration for TIM6 is due mainly to utilization of the intrinsic
binding energy of the nonreacting phosphate group of the
substrate.7
Scheme 2
Electrostatic interaction of the substrate phosphodianion with
TIM is essential for optimal substrate binding and enzymatic
activity.8 For example, the K12M active site mutation drastically
decreases the ability of TIM to bind both DHAP and its
monoanionic analogue dihydroxyacetone sulfate.8b However, there
has been no quantification of the contribution of the substrate
phosphodianion group to the enzymatic rate acceleration. The
activity of TIM toward isomerization of (R)-glyceraldehyde (GA)
to give dihydroxyacetone (DHA) would not be detected by
conventional enzyme assays, but we have found that the very
slow reaction of GA in the presence of a large amount of TIM
can be monitored by 1H NMR spectroscopy at 500 MHz. Figure
1 (b) shows the timecourse for the disappearance of 90% of 11
mM GA in the presence of 0.17 mM (4.5 mg/mL) rabbit muscle
TIM and 24 mM imidazole buffer (pD 7.0) in D2O at 25 °C and
I ) 0.1 (NaCl).9 1H NMR analysis showed that GA exists as
94.6% hydrate and 5.4% free carbonyl under these conditions
(Khyd ) 17.5) and the reaction was followed by monitoring the
disappearance of the C-1 proton of GA hydrate at 4.84 ppm (d,
J ) 5 Hz).11 The data give kobsd ) 4.6 × 10-6 s-1 for
disappearance of GA, which is 50-fold larger than kobsd ) 9.0 ×
10-8 s-1 determined in the absence of TIM under the same
conditions. Essentially all of the observed reaction of GA can be
attributed to the presence of the protein catalyst with kenz ) 4.5
1
× 10-6 s-1. Product analysis by H NMR showed that ca. 60%
of the products correspond to those of isomerization to give both
H-DHA and D-DHA and deuterium exchange into substrate to
give D-GA (Scheme 2). These product data will be discussed in
a full report.
Figure 1 (9) shows the timecourse for the disappearance of
11 mM GA in the presence of 0.17 mM (4.5 mg/mL) rabbit
muscle TIM, 18 mM imidazole buffer (pD 7.0), and 8 mM of
the potent competitive inhibitor 2-phosphoglycolate (PGA, Ki ≈
5 µM)12,13 in D2O at 25 °C and I ) 0.1 (NaCl). The data give
kobsd ) 1.6 × 10-6 s-1 for disappearance of GA, which is 10-fold
larger than kobsd ) 1.6 × 10-7 s-1 determined in the absence of
* Address correspondence to this author: (phone) (716) 645 6800 ext 2194;
(fax) (716) 645 6963; (e-mail) jrichard@chem.buffalo.edu.
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(9) (R)-Glyceraldehyde (80-90 wt % in H2O) from Aldrich was exchanged
with D2O before use. Rabbit Muscle TIM (lyophilized powder) from Sigma
was exhaustively dialyzed in the buffer of interest in D2O. The concentration
of TIM was determined from Vmax obtained in standard assays15 using a value
of kcat ) 2.6 × 105 min-1 for GAP.2,10 Periodic assay15 of the reaction mixture
(after 50 000-fold dilution) for TIM activity showed that there was no
significant loss of enzyme activity during the time period of these experiments
(up to one week).
(10) Krietsch, W. K. G.; Pentchev, P. G.; Klingenbuerg, H.; Hofstaetter,
T.; Buecher, T. Eur. J. Biochem. 1970, 14, 289-300.
(11) 1H NMR spectra at 500 MHz (64 transients) were acquired at 25 °C
using a Varian Unity Inova spectrometer with a 90° pulse angle, 6000 Hz
sweep width, 4 s acquisition time, and a 120 s relaxation delay.
(12) Wolfenden, R. Nature 1969, 223, 704-705.
(8) (a) Belasco, J. G.; Herlihy, J. M.; Knowles, J. R. Biochemistry 1978,
17, 2971-2978. (b) Lodi, P. J.; Chang, L. C.; Knowles, J. R.; Komives, E.
A. Biochemistry 1994, 33, 2809-2814. Joseph-McCarthy, D.; Lolis, E.;
Komives, E. A.; Petsko, G. A. Biochemistry 1994, 33, 2815-2823.
(13) Hartman, F. C.; LaMuraglia, G. M.; Tomozawa, Y.; Wolfenden, R.
Biochemistry 1975, 14, 5274-5279.
10.1021/ja016754a CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/17/2001