High impact information on FRAP1

• We demonstrate the utility of the enthalpy arrays by showing measurements for two protein-ligand binding interactions (RNase A + cytidine 2'-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler [1].
• Difference spectroscopic investigations on the interaction of brain hexokinase with glucose and glucose 6-phosphate (Glc-6-P) show that the binary complexes E-glucose and E-Glc-6-P give very similar UV difference spectra [2].
• Scatchard plots of the binding of glucose to brain hexokinase reveal only a single binding site but show distinct evidence of positive cooperativity, which is abolished by Glc-6-P and Pi [2].
• Brain hexokinase has no preexisting allosteric site for glucose 6-phosphate [2].
• P/2e- stoichiometries in six assay systems spanning different portions of the respiratory chain were estimated by direct determinations of Pi uptake in suspensions of bovine heart mitochondria containing a hexokinase trap [3].

Biological context of FRAP1

• Hexokinase (EC 2.7.1.1) catalyzes the first step in glucose metabolism, using ATP for the phosphorylation of glucose to glucose 6-phosphate [4].
• Direct binding studies of the interaction of Glc-6-P with brain hexokinase detect only a single high-affinity binding site for Glc-6-P (KD = 2.8 microM) [2].
• (b) At moderate flux, rate of hexokinase approached that of overall flux through the glycolytic pathway but "excessive" phosphofructokinase activity led to substrate cycling between fructose-6-PO4 and fructose 1,6-bisphosphate and resulted in a low net ATP yield (0-0.6 mol/mol of glucose) [5].
• An attempt was made to gain insight into the mechanism of orthophosphate attenuation of glucose-6-P inhibition of bovine brain hexokinase I (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from experiments of ligand binding and initial rate kinetics [6].
• The authors have chosen glycolysis as their starting point, concentrating on the regulatory enzymes, hexokinase, and, in a companion paper, phosphofructokinase [7].

Anatomical context of FRAP1

• Structure to function analysis identified an unduplicated, invariant N-terminal domain involved in HK1 outer mitochondrial membrane targeting, as well as putative carbohydrate and nucleotide-binding sites in the regulatory and catalytic halves of HK1 essential to enzyme function [8].
• Two products were generated from bovine cardiac muscle cDNA which show 82% nucleotide and 93% amino acid identity with a region of rat brain HK1 and cDNA [4].
• Hexokinase of calf trabecular meshwork [7].
• The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane [9].
• In addition, data are presented showing that addition of hexokinase plus glucose to submitochondrial particles in presence of ADP and Pi considerably lowers the Pi leads to HOH exchange but that further addition of cyanide or 2,4-dinitrophenol or both has little additional effect [10].

Associations of FRAP1 with chemical compounds

• The ATP-binding site in the catalytic half of the HK1 protein resembles nucleotide-binding regions from protein kinases, with the single amino acid replacement (lysine to glutamate) in the ATP-binding site of the amino half explaining the loss of HK1 catalytic function in the regulatory domain [8].
• A model is presented which is consistent with the binding and kinetic data currently available on the alleviation of glucose-6-P inhibition of brain hexokinase by phosphate [6].
• Studies on the mechanism of orthophosphate regulation of bovine brain hexokinase [6].
• Treatment of intact mitochondria with DCCD results in the inhibition of their ability to binding hexokinase (Nakashima, R. A., Mangan, P. S., Colombini, M., and Pedersen, P. L. (1986) Biochemistry 25, 1015-1021) [11].
• Hexokinase was increased in muscle alone whereas pyruvate kinase was significantly decreased in aorta [12].

Analytical, diagnostic and therapeutic context of FRAP1

• We have amplified and sequenced the complete coding region of bovine hexokinase isoenzyme 1 (HK1) from brain RNA with PCR primers selected for sequence conservation [8].
• The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC(50)=4.0+/-1.4 microM) [13].
• Microtubule-bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules [13].
• DL-propranolol in a concentration of 0.1 mM reduced HK activity in bovine lens epithelium after 72 hr in organ culture and disrupted lens light focusing ability after 250 hr of incubation [14].

References