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Function of your protein
The protein Ornithine Aminotransferase (OAT), in humans (hOAT), is an enzyme that catalyzes the the transfer of an amino group from L-ornithine(L-Orn) to α-ketoglutarate (α-KG). Its ligand, pyridoxal-5'-phosphate (), is a cofactor of this reaction. An amino group from L-Orn is transferred to PLP which converts it to pyridoxamine phosphate (PMP) and L-Orn is converted to L-glutamate-γ-semialdehyde. When the amino group of PMP is transferred to the α-KG, it forms glutamate and regenerates PLP.
Biological relevance and broader implications
hOAT is found in most tissues in the body but predominates in the liver and kidney. There is a need to study the enzyme hOAT because the overexpression of this protein aids the proliferation of cancer cells, specifically Hepatocellular carcinoma (HCC), a common form of liver cancer. hOAT has been a target for mechanism-based inactivators (MBIs) in ongoing drug design efforts. HCC is normally diagnosed at advanced stages where the tumors tend to be resistant to radiotherapy and chemotherapy, making this type of cancer difficult to treat.
In ongoing research, new hOAT inhibitors were created as fragmented-sized alternative substrates such as γ-aminobutyric acid () and 5-aminovaleric acid (). hOAT was soaked with GABA and AVA and the new substrates prevented original interactions with catalytic amino acids and the ligand PLP and provided a tighter binding to hOAT than L-ornithine. GABA covalently attached to PLP. While AVA covalently attached to PLP and Lysine 292, one of the catalytic enzymes in this binding pocket. GABA and AVA both displayed a stronger binding affinity and slower turnovers, making them strong demonstrators for potential drug targets of hOAT.
Important amino acids
Amino Acids in the are Lys 292, Asp 263, Arg 180. They are essential to the active site by providing certain . PLP is covalently bonded to the amino acid lysine. The nitrogen in the ring of PLP interacts with the negatively charged oxygen in the aspartate side chain. The phosphate group interacts with the positively charged nitrogen of the arginine side chain. Another interaction provided in this active site but not apart of the catalytic triad is the amino acid phenylalanine 177, there is a pi stacking interaction between its ring and the ring of the PLP. Other amino acids that have served importance to PLP and binding the substrate L-Orn appear to be . At a pH of 6.0 a salt bridge is formed between Arg 413 and Glu 235, these amino acids also regulate the width of the active site and establish the size of the channel that the substrate passes through. At a pH of 7.8 conformational changes of Arg 413 and Glu 235 causes the distance between the side chains to increase therefore that restriction of the active-site isn't as prevalent. Another conformational change occurs from a pH of 6.0 to 7.8 with Arg 180. At a pH of 6.0 Arg 180 is able to interact with PLP as previously described but that interaction is disrupted at a pH of 7.8. These conformational changes alter the binding affinity and contribute to a slower catalytic rate due to the decrease in interactions with less acidic conditions.
Structural highlights
hOAT is a protein with structures consisting of alpha helices, parallel and antiparallel beta sheets, and random coil. It's a polymer with a that has three subunits held together by non-covalent interactions like hydrogen bonds and salt bridges between the side chains of amino acids. Two of the subunits are bound and fit more closely together leaving a pocket/space between the two subunits shown in purple and orange and the subunit shown in green. Since there is an active within each subunit, the observed conformational changes seen in Arg 180, Arg 413, and Glu 235 that cause a decrease in reaction rate at a pH of 6.0 could also be contributed by the protonation state of other active sites within hOAT that don't induce conformational changes.
The of PLP are semi-exposed to the "outside world" but also slightly hidden into the protein. Having binding pockets exposed slightly can help other competitive substrates bind to this active site and inhibit the enzyme if necessary.
The ligand PLP has a phosphate group that is surrounded by other polar amino acids but the rest of the molecule, the carbons are surrounded by other non-polar amino acids to satisfy the needs of the . PLP would not be very stable if the polar/hydrophilic portions of the molecule were trying to interact with all surrounding non-polar/hydrophobic amino acids. This allows interactions such as hydrogen bonding and pi-stacking to stabilize and bind the ligand in the active site.
