Specific residues of the highly regulated fructose-1,6-bisphosphatase (FBPase) enzyme serve as important contributors to the catalytic activity of the enzyme. Previous clinical studies exploring the genetic basis of hypoglycemia revealed two significant mutations in the coding region of the FBPase gene in patients with hypoglycemia, linking the AMP-binding site to the active site of the enzyme. In the present study, a full kinetic analysis of similar mutants was performed. Kinetic results of mutants Y164A and M177A revealed an approximate two to three-fold decrease in inhibitory constants (Ki’s) for natural inhibitors AMP and fructose-2,6-bisphosphate (F2,6-BP) compared with the Wild-type enzyme (WT). A separate mutation (M248D) was performed in the active site of the enzyme to investigate whether the enzyme could be activated. This mutant displayed an approximate seven-fold increase in Ki for F2,6-BP. Interfacial mutants L56A and L73A exhibited an increase in Ki for F2,6-BP by approximately five-fold. Mutations in the AMP-binding site (K112A and Y113A) demonstrated an eight to nine-fold decrease in AMP inhibition. Additionally, mutant M248D displayed a four-fold decrease in its apparent Michelis constant (Km), and a six-fold increase in catalytic efficiency (CE). The importance—and medical relevance—of specific residues for FBPase structural/functional relationships in both the catalytic site and AMP-binding site is discussed.

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Human Fructose 1,6 Bisphosphatase (FBPase), an enzyme which serves as the control point in the gluconeogenesis pathway, was selected as a protein target for this study. FBPase is a putative target for drug development to combat Type II Diabetes. The FBPase protein structure has previously been solved (Zhang et al., 1995), and coordinates were readily available (PDB code IFTA) and downloaded for docking studies from the Rutgers Consortium of Structural Biology (RCSB) Protein Data Bank (PDB). The potential inhibitory molecules (IBS Natural Products Catalog) were downloaded from the ZINC Database (Irwin et al., 2012) in pdbqt file format. AutoDock Vina was selected as the ideal docking program for FBPase as determined by comparing co‐crystallized ligands to docked ligand positions. Selected compounds were chosen and docked into the FBPase active site and allosteric binding sites. Following the identification of promising theoretical binding constants for each molecule, molecular dynamics studies were performed for the FBPase protein target in the absence and presence of these promising molecules. Studies were caeried out using a NAMD/VMD (Nanoscale Molecular Dynamics with Visual Molecular Dynamics) system. NAMD implemented with CHARMM foce field is known to be highly efficient in simulating large systems for molecular motions. Dissociation constants (Ki) for the protein‐ligand complexes were calculated through NAMD runs, and respective conformational changes in the FBPase binding pockets were observed. Binding pockets were evaluated based on two factors: 1) pocket shape, and 2) pocket volume. Laboratory‐based site‐directed mutagenesis results revealed various levels of activation/inhibition of the enzyme as a result of mutations, an outcome with genetic implications for disease. Mutant enzymes resisted attempts at crystallization so molecular dynamics (MD) studies were performed on models of mutants. Focused on the active site of allosteric binding sites, further analysis was performed with key residues (virtually mutated) to initiate activation or inhibition of the enzyme (retention in T‐state or transition to R‐state) using NAMD. Resulting models of FBPase after MD runs shed light on possible interfacial structural changes produced in response to these mutations. Structural alterations in the models were observed in the active site, AMP (adenosine monophosphate) allosteric binding site, dimer interface allosteric binding site, and the interface between the active site and the AMP binding site. The signal from one part of the molecule is trasmittedto another part of the molecule through a known hydrogen‐bonding network (a sequential allosteric transition) vconnecting the active sites to the AMP allosteric binding sites. Novel hydrophobic networks were identified to connect the active sites to the allosteric binding sites using the NAMD protocol.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Monoamine oxidase (MAO) performs deamination of amines and is found bound to the outer mitochondrial membrane at high‐concentration in neuronal cells. There are two isoforms of MAO: MAO_A which oxidizes serotonin, noradrenaline and adrenaline, and MAO_B which oxidizes dopamine, b‐phenylethylamine (PEA), and benzylamine. Alterations in MAO activity can occur in some central and peripheral nervous system diseases. More specifically, heightened MAO_B activity in the brain occurs in Alzheimer's disease, Huntington's disease, Parkinson's disease and normal aging. Abnormal MAO_A activity has found to be associated with depression, anxiety and psychiatric disorders. Drugs have been developed and continue to be developed for both MAO_A and MAO_B as targets. MAO_A is inhibited by clorgyline and MAO_B is potently inhibited by both deprenyl and pargyline. Using these inhibitors as controls, a fluorescent activity assay was performed with commercially available catechins (green tea extracts), serotonin, and benzylamine substrates for MAO_A and MAO_B respectively, to investigate and confirm recent studies suggesting that green tea catechins (polyphenols) may be preventative for certain degenerative diseases and emotional illnesses utilizing MAOs as a target. Using a fluorescent assay, the Km value of serotonin to MAO_A was 1.75 μM and the Km value of benzylamine to MAO_B was 0.75 μM. The IC50 of clorgyline to MAO_A was 3.25 nM and that of deprenyl to MAO_B was 7.25 nM, in close agreement with the literature. The commercial catechins tested were found to have IC50s in the low‐to‐mid μM range (~50–750 μM). Efforts to purify catechins are underway to repeat these studies. Molecular docking of specific catechins into the MAO_A and MAO_B active sites resulted in binding constants in the low μM range (in agreement with experimentally determined Km values for natural substrates). Crystallization studies of MAO/catechin complexes are in progress.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.