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The functional role of the conserved active
site proline of triosephosphate isomerase
1#§
Casteleijn ,
2§
Alahuhta ,
1
Groebel ,
3
El-Sayed ,
Marco G.
Markus
Katrin
Ibrahim
3
4
1
2
Koen Augustijns , Annemie Lambeir , Peter Neubauer , Rik Wierenga .
1) University of Oulu (Finland), Bioprocess Engineering Laboratory, Dept. of Process and Environm. Engineering and Biocenter Oulu; 2)
University of Oulu (Finland), Dept. of Biochemistry and Biocenter Oulu; 3) University of Antwerp (Belgium), Laboratory of Medicinal
Chemistry; 4). University of Antwerp (Belgium), Laboratory of Medicinal Biochemistry. §These authors contributed equally to this work; #
corresponding author: [email protected].
Introduction
Figure 1
TIM catalyses the interconversion
of an α-hydroxyketone (DHAP)
and a chiral α-hydroxyaldehyde
(DGAP). The covalent structure of
the transition state analogue 2phosophoglycollate (2PG) is also
shown.
Triose phosphate isomerase (TIM) is a glycolytic enzyme with very high
substrate specificity. TIM catalyses the interconversion of Dglyceraldehyde-3-phosphate (DGAP) and dihydroxyacteone phosphate
(DHAP), as seen in Figure 1. The wild type enzyme is a dimer, and each
subunit has the classical TIM-barrel fold.
Our studies are focusing on understanding the catalytic properties of the
Wild Type (Wt-TIM) [1, 2] and applying this knowledge to our newly
engineered enzymes.
Enzymology of P168A vs. wild type
Structural information
Wild Type TIM is well studied, and many publications can be found on its
catalytic and structural properties. However, some aspects are still poorly
understood. Observations in a liganded structure of Leishmania TIM
showed P168 (N-terminal hinge) in a planar state which is a strained[2]
conformation, which could facilitate loop opening. Furthermore, proline
168 is fully conserved in all TIM species.
Structural investigation of liganded (with 2PG) and unliganded P168A
mutant revealed two new observations. As seen in figure 3A, loop-6 and
loop-7 are in the closed conformation as in wild type. However, the
conformational switch from “swung out” to “swung in” of the catalytic
glutamate (Glu167) has not occurred upon ligand binding in the P168A
mutant. This clearly affects the mode of binding of the transition state
analogue were the mode of binding is “down”; unlike in wild type (Figure 3B).
We investigated the enzymological properties of wild type TIM and its
P168A mutant regarding the loop-6/loop-7 mechanism. The results are
summerized in figure 2, where the impact on conversions per second and
affinity for 2PG (see also Figure 1) can be seen. Interestingly the affinity
for the phosphate moiety of the substrate (see figure 1) is not affected.
The results clearly show the importance of proline 168 for catalytic
activity.
A
This investigation, and of others [3, 4, 5], are of importance for protein
engineering efforts on TIM, since only in the closed form the active site is
competent for catalysis. This knowledge can then be applied to engineer
(more) active monomeric enzyme variants (based on ml8b TIM and our
own engineered A-TIM) regarding poorly binding (new) ligands.
Figure 3
A) Comparison between the
liganded P168A (grey) and wild
type TIM structures (purple). The
dotted lines indicate the hydrogen
bonding
interactions
between
Glu167 and Ser96. Loop 6 is
closed in both structures.
B) Detail of the mode of binding of
2PG in wild type (green) and the
P168A mutant (blue). The clash of
O (Gly211) with Pro168 induced by
ligand binding is indicated by an
arrow.
Both pictures were made with ICM
(www.molsoft.com).
B
The second new observation is that the
trigger for loop-6 closure is induced by the
interactions of the phosphate moiety of the
ligand with residues in loop-7.
This switch of loop-6 is facilitated, or even
triggered, by the movement of O(Gly211) in loop-7 as it clashes with
CD(Pro168). This occurs if the Pro168 side chain would not move on ligand
binding (Fig. 3B).
This switch does not exist in the P168A mutant.
Conclusions
Figure 2
Enzymology studies on P168A mutant versus wild type. Left the dramatic drop in relative Kcat
over Km can be seen. The right graph shows the affinity decrease for the transition state
analogue 2PG.
► Proline 168 is needed for conformational strain around the catalytic
glutamate. This strain is transferred to the catalytic glutamate.
► Ligand binding triggers the conformational switch of the catalytic
machinery needed for catalysis.
References
Acknowledgements
1. Kursula, I., and Wierenga, R.K. (2003) Crystal structure of triosephosphate isomerase complexed with 2-phospho-glycolate at 0.83Å resolution. J. Biol. Chem 278, 9544-9551.
2. Kursula, I., Salin, M., Sun, J., Norledge, B.V., Haapalainen, A.M., Sampson, N.S., and Wierenga, R.K. (2004) Understan-ding protein lids: structural analysis of active hinge mutants in
triosephosphate isomerase. PEDS (submitted). (about importance of A178L)
3. Norledge, B.V., Lambeir, A.M., Abagyan, R.A., Rottmann, A., Fernandez, A. M., Filimonov, V.V., Peter, M.G., Wierenga, R.K. 2001. Modelling, mutagenesis and structural studies on the fully
conserved phosphate binding loop (loop-8) of triosephosphate isomerase: towards a new substrate specificity. Proteins 42, 383-389.
4. Schliebs, W. Thanki, N., Eitja, R., Wierenga, R.W. (1996) Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural studies. Prot.
Science 5, 229-239.
5. Thanki, N., Zeelen, J.Ph., Mathieu, M, Jaenicke, R, Abagyan, R.A.A., Wierenga, R.K., Schliebs, W. (1997) Protein engineering with monomeric triophosphate isomerase (monoTIM): the
modelling and structure verification of a seven-residue loop. Protein Engin. 10(2), 159-167.
This work is supported by the Academy of
Finland (project 53923) an Tekes project
“Biocatnuc”
We have much appreciated the help from
the staff at the beam-line BW7A
(EMBL/DESY in Hamburg) for collecting
the data of the liganded P168A structure.
The skillful technical support from Ville
Ratas has been invaluable in this work.