Loop-6 and loop-7 of triosephosphate isomerase: structural enzymological studies.
Rik Wierenga, Markus Alahuhta, Marco Casteleijn, Peter Neubauer

Wierenga, R.K.1, Alahuhta, M.P.1, Casteleijn, M.1,2, Neubauer, P.1,2, Biocenter Oulu1, Departments of Biochemistry1 and Engineering2, University of Oulu, Linnanmaa, FIN-90570, Oulu, Finland.

In triosephosphate isomerase (TIM) the conformational switches of the active site loops (loop-6 and loop-7) are correlated. Ligand binding induces small main chain conformational changes of the Y210GGS motif in loop-7 and a 7Å closure movement of the tip of loop-6. Classically loop-6 has been described of consisting of 11 residues, P168VWAIGTGKTA (Kursula et al, (2004) PEDS, 17, 375-382), but it has been noted that small but significant changes of the main chain of Glu167, just before loop-6, can be observed. Also, the side chain of the catalytic glutamate moves from a swung-out into a swung-in position, competent for catalysis. The proline residue of Pro168 is fully conserved in 133 TIM-sequences. In our studies aimed at better understanding of the wild type TIM catalytic properties we have mutated Pro168 into Ala168. Kinetic and structural properties of the P168A variant have been determined. In the liganded P168A structure loop-6 and loop-7 have adopted the closed conformation. These studies show two possible functions for a proline at this position, all related to the unique property of the pyrrolidine ring system of this side chain: (i). The P168A mutation generates a free NH-group near the active site. It is observed in the new structure of the P168A liganded structure, that this NH-group is hydrogen bonded to O(Gly211) of the closed loop-7 conformation, there by stabilizing this closed conformation which probably makes it more difficult for the product to leave at the end of the reaction cycle. (ii) In the liganded P168A structure, unexpectedly, the main chain and side chain of the catalytic glutamate are seen in the open, swung-out conformation, previously only seen in unliganded structures: apparently, the conformational switches of loop-6 and loop-7 (stabilized by the phosphate moiety of the ligand) is not transmitted to the catalytic glutamate. This observation suggests that the rigid proline side chain geometry is required for transmitting the conformational switch of loop-6 and loop-7 to the catalytic glutamate.
The implications of these observations for our work on changing the substrate specificity of monomeric variants of TIM will be discussed.