PubMed · 9768349
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
F McCormick. 1998-09-24. Going for the GAP.. https://doi.org/10.1016/s0960-9822(98)70431-2
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
We present a novel method designed to analyze the discriminative ability of knowledge-based potentials with respect to the 20 residue types. The method is based on the preference of amino acids for specific types of protein environment, and uses a virtual mutagenesis experiment to estimate how much information a given potential can provide about environments of each amino acid type. This allows one to test and optimize the performance of real potentials at the level of individual amino acids, using actual data on residue environments from a dataset of known protein structures. We have applied our method to long-range and medium-range pairwise distance-dependent potentials. The results of our study indicate that these potentials are only able to discriminate between a very limited number of residue types, and that discriminative ability is extremely sensitive to the choice of parameters used to construct the potentials, and even to the size of the training dataset. We also show that different types of pairwise distance potentials are dominated by different types of interactions. These dominant interactions strongly depend on the type of approximation used to define residue position. For each potential, our methodology is able to identify a potential-specific amino acid distance matrix and a reduced amino acid alphabet of any specified size, which may have implications for sequence alignment and multibody models.
Glutamate transporters play an important role in homeostasis of extracellular glutamate, a major excitatory neurotransmitter and a potential neurotoxin. In mammalian brain, glutamate transporter type 2 (EAAT2) is the most abundant form. Studies of molecular structures demonstrated that tyrosine 403 is critical in regulating the ion selectivity and transport mode of EAAT2. We hypothesized that wild type EAAT2 and its mutant at tyrosine 403 have different responses to volatile anesthetics, commonly used anesthetics that have been shown to affect glutamate transporter activity and decrease extracellular glutamate concentrations. We used site-directed mutagenesis and oocyte expression systems to test the hypothesis. Volatile anesthetics did not affect the activity of wild type EAAT2, isolated from rat hippocampus. When tyrosine 403 was replaced by histidine (Y403H), volatile anesthetics (isoflurane or halothane) at clinically relevant concentrations significantly decreased the transporter activity. Okadaic acid, a phosphatase inhibitor, significantly prolonged the isoflurane-induced inhibition. This inhibition was reversed by staurosporine and calphostin C, two protein kinase C (PKC) inhibitors, but not by the third PKC inhibitor, chelerythrine. Phorbol 12-myristate 13-acetate, a PKC activator, inhibited the activity of both wild type and Y403H EAAT2. This inhibition was also reversed by the same two PKC inhibitors but not by the third one. These results suggest that the switch of tyrosine 403 to histidine rendered EAAT2 sensitive to volatile anesthetics, a phenomenon that may require protein phosphorylation. PKC may be involved in the regulation of the activity of both wild type and Y403H EAAT2.
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.