Evolution of Metal Ion and Purine Nucleotide Effects and Cooperativity in Glutamate Dehydrogenase: The Role of Conformational Flexibility

Higher eukaryote glutamate dehydrogenases are highly allosterically regulated by a wide variety of compounds including purine nucleotides and metal ions. Of the purine nucleotides ADP and GTP are the most potent allosteric heterotropic effectors while NAD(P)+ shows negative homotropic allosteric effects involving communication between active sites. To investigate potential evolutionary factors we have compared a variety of available structures and homology models of glutamate dehydrogenases that either show allosteric effects or lack allosteric interactions to elucidate the formation of either the purine heterotropic sites or the metal binding sites of the enzyme using HINT computational analysis of contributions to binding. Local Spatial Pattern alignments have identified potential conformationally flexible spines that could form pathways of conformational changes. The effects of purine nucleotides and various metal ions on overall stability have been determined by both heat and chemical denaturation using CD and fluorescence spectroscopy. Inhibition is correlated with decreased flexibility while activators enhance flexibility. Similar studies with inhibitory or non‐inhibitory meal ions and limited proteolysis experiments using immobilized trypsin and tandem mass spectrometry to identify initial cleavage sites provide more detailed spatial information are in progress.