Human Glutamate Oxaloacetate Transaminase Research Articles

Dimerization misalignment in human glutamate-oxaloacetate transaminase variants is the primary factor for PLP release.

The active form of vitamin B6, pyridoxal 5’-phosphate (PLP), plays an essential role in the catalytic mechanism of various proteins, including human glutamate-oxaloacetate transaminase (hGOT1), an important enzyme in amino acid metabolism. A recent molecular and genetic study showed that the E266K, R267H, and P300L substitutions in aspartate aminotransferase, the Arabidopsis analog of hGOT1, genetically suppress a developmentally arrested Arabidopsis RUS mutant. Furthermore, CD analyses suggested that the variants exist as apo proteins and implicated a possible role of PLP in the regulation of PLP homeostasis and metabolic pathways. In this work, we assessed the stability of PLP bound to hGOT1 for the three variant and wildtype (WT) proteins using a combined 6 μs of molecular dynamics (MD) simulation. For the variants and WT in the holo form, the MD simulations reproduced the “closed-open” transition needed for substrate binding. This conformational transition was associated with the rearrangement of the P15-R32 small domain loop providing substrate access to the R387/R293 binding motif. We also showed that formation of the dimer interface is essential for PLP affinity to the active site. The position of PLP in the WT binding site was stabilized by a unique hydrogen bond network of the phosphate binding cup, which placed the cofactor for formation of the covalent Schiff base linkage with K259 for catalysis. The amino acid substitutions at positions 266, 267, and 300 reduced the structural correlation between PLP and the protein active site and/or integrity of the dimer interface. Principal component analysis and energy decomposition clearly suggested dimer misalignment and dissociation for the three variants tested in our work. The low affinity of PLP in the hGOT1 variants observed in our computational work provided structural rationale for the possible role of vitamin B6 in regulating metabolic pathways.

Expression, purification and preliminary crystallographic studies of human glutamate oxaloacetate transaminase 1 (GOT1)

Glutamate oxaloacetate transaminase (GOT) catalyzes the reversible reaction of l-aspartate and α-ketoglutarate into oxaloacetate and l-glutamate and plays a key role in carbon and nitrogen metabolism in all organisms. In human tissues, GOTs are pyridoxal 5′-phosphate-dependent (PLP) enzymes which exist in cytoplasm and mitochondrial forms, GOT1 and GOT2, respectively. GOT1 expression correlates with the growth of several tumors because cancer cells can utilize the amino acid glutamine to fuel anabolic processes, and therefore, GOT1 represents a new therapeutic target in cancer. In this work, human GOT1 was expressed in Escherichia coli periplasmic space, and purified by a combination of His-tag immobilized metal-ion affinity chromatography and anion exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 37°C and a pH of 7.5. Cations such as Na+, K+ and Mg2+ slightly inhibited the activity of recombinant human GOT1, while Zn2+, Mn2+, Cu2+, Ni2+, Co2+ and Ca2+ had stronger inhibitory effects. Crystals of human GOT1 were grown using the hanging-drop vapor diffusion method at 4°C with 0.1M Bis–Tris pH 6.0% and 21% (w/v) PEG 3350. The crystals diffracted to 2.99Å resolution and belonged to space group P43212 with the unit cell parameters a=b=93.4, c=107.4Å, α=β=γ=90°.

Pharmacokinetics of Glutamate–Oxaloacetate Transaminase and Glutamate–Pyruvate Transaminase and Their Blood Glutamate-Lowering Activity in Naïve Rats

Traumatic brain injury (TBI) and stroke lead to elevated levels of glutamate in the brain that negatively affect the neurological outcomes in both animals and humans. Intravenous administration of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) enzymes can be used to lower the blood glutamate levels and to improve the neurological outcome following TBI and stroke. The objective of this study was to analyze the pharmacokinetics and to determine the glutamate-lowering effects of GOT and GPT enzymes in naïve rats. We determined the time course of serum GOT, GPT, and glutamate levels following a single intravenous administration of two different doses of each one of the studied enzymes. Forty-six male rats were randomly assigned into one of 5 treatment groups: saline (control), human GOT at dose 0.03 and 0.06 mg/kg and porcine GPT at dose 0.6 and 1.2 mg/kg. Blood samples were collected at baseline, 5 min, and 2, 4, 8, 12, and 24 h after the drug injection and GOT, GPT and glutamate levels were determined. The pharmacokinetics of both GOT and GPT followed one-compartment model, and both enzymes exhibited substantial glutamate-lowering effects following intravenous administration. Analysis of the pharmacokinetic data indicated that both enzymes were distributed predominantly in the blood (central circulation) and did not permeate to the peripheral organs and tissues. Several-hour delay was present between the time course of the enzyme levels and the glutamate-lowering effects (leading to clock-wise hysteresis on concentration-effect curves), apparently due to the time that is required to affect the pool of serum glutamate. We conclude that the interaction between the systemically-administered enzymes (GOT and GPT) and the glutamate takes place in the central circulation. Thus, glutamate-lowering effects of GOT and GPT apparently lead to redistribution of the excess glutamate from the brain's extracellular fluid into the blood and can reduce secondary brain injury due to glutamate neurotoxicity. The outcomes of this study regarding the pharmacokinetic and pharmacodynamic properties of the GOT and GPT enzymes will be subsequently verified in clinical studies that can lead to design of effective neuroprotective treatment strategies in patients with traumatic brain diseases and stroke.

Genetic polymorphism of human glutamate oxaloacetate transaminase (GOT1) detected using isoelectric focusing and a sensitive and positive staining method

Genetic polymorphism of glutamate oxaloacetate transaminase (GOT1) was demonstrated in human erythrocytes by isoelectric focusing in thin layer polyacrylamide gels and a sensitive and positive detection method. Using this technique, five phenotypes, GOT1 1, 2, 2-1, 3-1 and 3-2 were determined and the estimated gene frequencies of GOT1*1, GOT1*2 and GOT1*3 in the Japanese population were 0.9740, 0.0173 and 0.0087, respectively.

Synteny between the pro+ marker and human glutamate oxaloacetate transaminase.

Chinese hamster ovary cells with a specific auxotrophy for proline were fused with human cells from a variety of sources and the resulting hybrids analyzed for human genetic markers. Of 63 hybrid clones examined, 27 possessed both proline and cytoplasmic glutamate oxaloacetate transaminase markers; 36 had neither; and no clones were found possessing one and not the other. These results constitute evidence that the proline and glutamate oxalocetate transaminase markers are syntenic. Evidence for absence of synteny between these and a variety of other human genes is presented. Biochemical tracer experiments established that the proline biosynthetic pathway through glutamate has been restored in the Pro+ hybrids.