3 NEW CRYSTAL-STRUCTURES OF POINT MUTATION VARIANTS OF MONOTIM - CONFORMATIONAL FLEXIBILITY OF LOOP-1, LOOP-4 AND LOOP-8

Background: Wild-type triosephosphate isomerase (TIM) is a very stable dimeric enzyme. This dimer can be converted into a stable monomeric p rotein (monoTIM) by replacing the 15-residue interface loop (loop-3) b y a shorter, 8-residue, loop. The crystal structure of monoTIM shows t hat two active-site loops (loop-1 and loop-4), which are at the dimer interface in wild-type TIM, have acquired rather different structural properties. Nevertheless, monoTIM has residual catalytic activity. Res ults: Three new structures of variants of monoTIM are presented, a dou ble-point mutant crystallized in the presence and absence of bound inh ibitor, and a single-point mutant in the presence of a different inhib itor. These new structures show large structural variability for the a ctive-site loops, loop-1, loop-4 and loop-8. In the structures with in hibitor bound, the catalytic lysine (Lys13 in loop-1) and the catalyti c histidine (His95 in loop-4) adopt conformations similar to those obs erved in wild-type TIM, but very different from the monoTIM structure. Conclusions: The residual catalytic activity of monoTIM can now be ra tionalized. In the presence of substrate analogues the active-site loo ps, loop-1, loop-4 and loop-8, as well as the catalytic residues, adop t conformations similar to those seen in the wild-type protein. These loops lack conformational flexibility in wild-type TIM. The data sugge st that the rigidity of these loops in wildtype TIM, resulting from su bunit-subunit contacts at the dimer interface, is important for optima l catalysis.