IS A HYDROPHOBIC AMINO-ACID REQUIRED TO MAINTAIN THE REACTIVE V-CONFORMATION OF THIAMIN AT THE ACTIVE-CENTER OF THIAMIN DIPHOSPHATE-REQUIRING ENZYMES - EXPERIMENTAL AND COMPUTATIONAL STUDIES OF ISOLEUCINE-415 OF YEAST PYRUVATE DECARBOXYLASE

The residue I415 in pyruvate decarboxylase from Saccharomyces cerevisi ae was substituted with a variety of uncharged side chains of varying steric requirements to test the hypothesis that this residue is respon sible for supporting the V coenzyme conformation reported for this enz yme [Arjunan et al, (1996) J. Mol. Biol, 256, 590-600], Changing the i soleucine to valine and threonine decreased the k(cat) value and shift ed the k(cat)-pH profile to more alkaline values progressively, indica ting that the residue at position. 415 not only is important for provi ding the optimal transition state stabilization but also ensures corre ct alignment of the ionizable groups participating in catalysis. Subst itutions to methionine (the residue used in pyruvate oxidase for this purpose) or leucine (the corresponding residue in transketolase) led t o greatly diminished k(cat) values, showing that for each thiamin diph osphate-dependent enzyme an optimal hydrophobic side chain evolved to occupy this key position. Computational studies were carried out on th e wild-type enzyme and the I415V, I415G, and I415A variants in both th e absence and the presence of pyruvate covalently bound to C2 of the t hiazolium ring (the latter is a model for the decarboxylation transiti on state) to determine whether the size of the side chain is criticall y required to maintain the V conformation. Briefly, there are sufficie nt conformational constraints from the binding of the diphosphate side chain and three conserved hydrogen bonds to the 4'-aminopyrimidine ri ng to enforce the V conformation, even in the absence of a large side chain at position 415, There appears to be increased coenzyme flexibil ity on substitution of Ile415 to Gly in the absence compared with the presence of bound pyruvate, suggesting that entropy contributes to the rate acceleration. The additional CH3 group in Ile compared to Val al so provides increased hydrophobicity at the active center, likely cont ributing to the rate acceleration. The computational studies suggest t hat direct proton transfer to the 4'-imino nitrogen from the thiazoliu m C2H is eminently plausible.