Decarboxylase Structure-Function Relationships

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A structural feature common to apoproteins of all pyridoxal 5'-phosphate (PLP) dependent carboxylases is an active site lysine which forms a Schiff base between the epsilon amino group and the aldehyde moiety of the cofactor. Dialkyl glycine decarboxylase (DGD) from Pseudomonas cepacia is probably such an enzyme. It can catalyze both decarboxylation and transamination reactions. Previous work with this enzyme suggests that the proton transfer is unusual. DGD catalyzes proton transfer to the 4' carbon of the cofactor instead of the alpha carbon of the amino acid. A reasonable hypothesis is that DGD is structurally similar to other decarboxylases but differs from them by inverting the basicities of histidine and lysine. This change would place the solvent proton preferentially on lysine which is closer to the cofactor 4' carbon. In order to test the validity of this model, mutants will be constucted with alanine substituting for the lysine at the active site. If the experimental outcome confirms this hypothesis, it will tell us a good deal about the electric field strengths at the active site and how such local fields affect the free energy contents of enzyme-substrates complexes and transition states. Dr. Keller is conversant with the enzyme and has been well trained. The project would have a fovarable impact on the quality of undergraduate education at the host institution.