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 cerevisiae was substituted with a variety of uncharged side chains of varying steric requirements to test the hypothesis that this residue is responsible for supporting the V coenzyme conformation reported for this enzyme [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. Changing the isoleucine to valine and threonine decreased the kcat value and shifted the kcat-pH profile to more alkaline values progressively, indicating that the residue at position 415 not only is important for providing the optimal transition state stabilization but also ensures correct alignment of the ionizable groups participating in catalysis. Substitutions to methionine (the residue used in pyruvate oxidase for this purpose) or leucine (the corresponding residue in transketolase) led to greatly diminished kcat values, showing that for each thiamin diphosphate-dependent enzyme an optimal hydrophobic side chain evolved to occupy this key position. Computational studies were carried out on the wild-type enzyme and the I415V, I415G, and I415A variants in both the absence and the presence of pyruvate covalently bound to C2 of the thiazolium ring (the latter is a model for the decarboxylation transition state) to determine whether the size of the side chain is critically required to maintain the V conformation. Briefly, there are sufficient conformational constraints from the binding of the diphosphate side chain and three conserved hydrogen bonds to the 4'-aminopyrimidine ring to enforce the V conformation, even in the absence of a large side chain at position 415. There appears to be increased coenzyme flexibility 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 also provides increased hydrophobicity at the active center, likely contributing to the rate acceleration. The computational studies suggest that direct proton transfer to the 4'-imino nitrogen from the thiazolium C2H is eminently plausible.
Guo
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F
Department of Chemistry, Program in Cellular and Molecular Biodynamics, Rutgers, the State University of New Jersey 07102, USA.
Zhang
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D
Kahyaoglu
A
A
Farid
R S
RS
Jordan
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F
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D019295:Computational Biology / Q000379:methods; D007532:Isoleucine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007532:Isoleucine; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate