Kinetic and crystallographic studies of the Schiff-base enzymes acetoacetate decarboxylase and fructose-1,6-(bis)phosphate aldolase

The Schiff-base enzymes acetoacetate decarboxylase and fructose-1,6-(bis)phosphate aldolase make use of both covalent and acid/base catalysis during the course of catalyzing their reactions. Acetoacetate decarboxylase catalyzes the breakdown of acetoacetic acid to CO2 and acetone, while fructose-1,6-bisphosphate aldolase catalyzes the reversible breakdown of fructose-1,6-(bis)phosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate in glycolysis. This work used the complimentary tools of enzyme kinetics and x-ray crystallography to analyse the chemical mechanisms utilized by these enzymes. Acetoacetate decarboxylase was probed with substrate analogs and inhibitors to determine the flexibility of the active site. Proton exchange was measured using 1D NMR to determine whether enamine formation was a rate-limiting step. The protein was crystallized, and 14 complete data sets collected of unliganded, selenomethionine substitute and metal-bound complex. However, phases could not be obtained to solve the structure as the crystals were twinned by merohedry. Fructose-1,6-bisphosphate aldolase A (FBP-A) was crystallized in the presence of IP3, a putative inhibitor of FBP-A, to determine if a third phosphate binding site exists. The structure of FBP-A revealed a phosphate bound in the active along with a novel structure for the C-terminal region. Comparison of this structure with other FBP isozymes has yielded some insights into the ways the C-terminal isozyme specific residues (ISRs) determine the kinetic characteristics of each isoform. Specifically this structure has revealed that the C-terminal tyrosine may move from the P 1-binding site during binding of substrate. Also, the location of the "trigger" residue Arg303 was compared to other structures to generate a model of its function. Finally, FBP-A and AADase are compared to examine the "Westheimer Hypothesis", which suggests that the positive charge of one lysine in the active site keeps the schiff-base lysine deprotonated and available for covalent catalysis, in the context of each enzymes functional active-site groups.