Study of Soluble Glucose Dehydrogenase (sGDH) based upon Kinetic Studies of the Structural Analogs of PQQ. James Ewing, Carolnn Yong & Leslie Thompson
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Soluble glucose dehydrogenase is a homodimeric enzyme found in various bacteria such as Acinetobacter calcoaceticus , requiring Ca<sup>2+</sup> and pyrroloquinolinequinone (PQQ) cofactors for its full enzymatic activity. sGDH catalyses the oxidation of glucose and other aldose sugars. The mechanistic details have yet to be completely disclosed. Here, three isomeric analogs of PQQ are systematically selected and synthesized from methoxynitroaniline derivatives. Each synthesis includes the reaction of the diazonium salt with ethyl 2-methylacetoacetate, yielding the corresponding substituted phenylhydrazone of ethyl pyruvate.Then, each hydrazone underwent acid catalyzed Fischer indole synthesis, followed by reduction of the nitro group using H2/Pd/C to the corresponding aminoindole. Michael addition of an aminoindole derivative with dimethyl trans-2-ketoglutaconate was performed to form the pyridine ring, which was further oxidized into the triester of a PQQ analog. Hydrolysis of the oxidation product yields an isomeric analog of PQQ. These isomers differ in the positioning of the nitrogen atom on the ring system and will serve to define the structural significance of PQQ in terms of binding with a metal cation and reconstitution process with the apoprotein. Detailed analysis of the stability of each reconstituted enzyme as well as steady-state kinetics studies will shed light on the structural characteristics mandatory for enzymatic activity, substrate specificity, and active site characterization.