RESUMEN
The kinetic properties of fatty acid synthetase, extracted from the mycelia of Neurospora crassa, were studied to determine the role of the multi-enzyme complex in the regulation of long chain saturated fatty acid synthesis. Acetyl-CoA, a substrate, affects the fatty acid synthetase with increasing concentration in a normal Michaelis-Menton kinetic mode. Malony-Coa, another substrate, activates the synthetase in a homotropic manner up to 35 microM with a Hill coefficient of 2.8. Above that concentration a sudden reverse trend in its effect takes over, then a gradual decrease in enzyme activity is observed with increasing substrate concentration. Citrate activates the synthetase to a maximum at 1 mM of citrate then reverses its effect and levels off at 2 mM. At the higher citrate concentrations the multi-enzyme complex has an activity twice that in the absence of citrate. Palmityl-CoA in low concentrations inhibits the synthetase. The inhibition is suppressed by the addition of bovine serum albumin. Increasing the concentration of the albumin enhances the suppression but does not eliminate the inhibition. These findings indicate that fatty acid synthetase could indeed have a key regulatory role.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Neurospora crassa/enzimología , Neurospora/enzimología , Acetilcoenzima A/farmacología , Citratos/farmacología , Ácido Graso Sintasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , Malonil Coenzima A/farmacología , Manganeso/farmacología , Palmitoil Coenzima A/farmacologíaRESUMEN
The kinetic properties of pyruvate kinase (EC 2.7.1.40) extracted from the mycelia of Neurospora crassa were examined at physiological pH to determine the role of the enzyme in the regulation of glycolysis. The velocity curve with the substrates, phosphoenolpyruvate and adenosine diphosphate, are hyperbolic. The effect of magnesium, potassium, or calcium on the enzyme is influenced by the pH but not to the extent that would change their role as cofactor or inhibitor. Adenosine triphosphate and citrate remain strong inhibitors even with changes in pH. Fructose-1,6-diphosphate and glucose-6-phosphate are the dual positive effectors at physiological pH. Valine is the only amino acid that inhibits the enzyme at a concentration range of valine found in the mycelial juice. Thus, the properties of the enzyme at physiological pH are significantly different from those observed at neutral pH of the usual assay conditions, but its role as a key regulator of glycolysis is unchanged.
Asunto(s)
Neurospora crassa/enzimología , Neurospora/enzimología , Piruvato Quinasa/metabolismo , Adenosina Trifosfato/farmacología , Aminoácidos/análisis , Calcio/farmacología , Activación Enzimática/efectos de los fármacos , Fructosadifosfatos/farmacología , Glucofosfatos/farmacología , Concentración de Iones de Hidrógeno , Cinética , Magnesio/análisis , Neurospora crassa/análisis , Potasio/análisis , Piruvato Quinasa/antagonistas & inhibidoresRESUMEN
Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40), extracted from the mycelium of Neurospora crassa has been purified 560-fold by precipitation with ammonium sulphate, chromatography with DEAE-Sephadex, and gel filtration with Sephadex G-200. Potassium and magnesium are required for enzyme activity. Fructose, 1,6-diphosphate is the only physiological activator found for the enzyme. In decreasing order of potency, citrate, oxalacetate, calcium, and ATP are inhibitors. Phosphoenolpyruvate is cooperatively bound by the enzyme and the cooperatively is reduced by ATP and completely eliminated by fructose-1,6-diphosphate. Lowering of pH from 7-5 to 5-5 changes the Hill coefficient from 2-7 to 1-0. Substitution of ADP by other nucleotides reduces enzyme activity. Manganese can substitute for the cofactor magnesium, but the reaction velocity is then reduced. MgADP- is cooperatively bound by the enzyme and inhibition of the enzyme occurs only when either magnesium or ADP is in excess of the other beyond the optimum concentration. These kinetics properties of pyruvate kinase are compatible with the role of a regulator of glycolysis in Neurospora crassa.