Sequential feedback inhibition and regulation of liver carbohydrate metabolism through control of enzyme activity.

Abstract 1. (1)|The free fatty acids, octanoic, lauric, myristic, palmitic, and elaidic acids, were examined for their ability to inhibit the key glycolytic enzymes. The effective inhibitory concentrations of the FFA were in the physiological range of rat or human systems. 2. (2)|Acetyl-CoA was observed to exert a dose- and preincubation time-dependent inhibitory action on hepatic glucokinase and pyruvate kinase. When acetyl-CoA was added to the reaction mixture of liver pyruvate kinase assay system a K i = 5.1 m m was found, but when it was added to the supernantant fluid for a 10-min preincubation a K i = 30 μ m was observed. The inhibition was not of a competitive type. Acetyl-CoA required only a few seconds for the interaction resulting in the inhibition of liver pyruvate kinase. 3. (3)|Acetyl-CoA also inhibited rat muscle pyruvate kinase with a K i = 2.0 × 10 −4 m , and the inhibition was of a non-competitive type. 4. (4)|Acetyl-CoA inhibited glucokinase, in a preincubation system, yielding a K i = 75 μ m . The inhibition of this enzyme was through a non-competitive mechanism. 5. (5)|There was no inhibition observed with acetyl-CoA against liver hexokinase, phosphofructokinase, lactate dehydrogenase, glucose 6-phosphatase or fructose 1,6-diphosphatase. Yeast glucose 6-phosphate dehydrogenase and rabbit muscle lactate dehydrogenase were also unaffected by acetyl-CoA. 6. (6)|FFA which is an end product of glucose catabolism acts as a feedback inhibitor by inhibiting hexokinase, glucokinase, phosphofructokinase, and pyruvate kinase. Acetyl-CoA which is an end product of the degradation of FFA reconfirms the feedback inhibition by inhibiting glucokinase and pyruvate kinase activities. This phenomenon was called sequential feedback inhibition. 7. (7)|Phosphoenolpyruvate when preincubated for 10 min with liver supernatant fraction caused a progressive inhibition of glucokinase activity with a K i = 2.1 m m . Kinetic experiments showed that the Km for glucokinase was not affected and the inhibition was not of a competitive type. Liver hexokinase was not inhibited by phosphoenolpyruvate. 8. (8)| l -alanine exerted a concentration-dependent inhibition of liver pyruvate kinase activity which was detectable at low concentrations of PEP. At the usual PEP concentration employed in the routine pyruvate kinase assay system l -alanine was not inhibitory with or without preincubation. At low concentrations of PEP l -alanine acted as a competitive inhibitor against PEP. Using a preincubation mixture it was observed that alanine protected pyruvate kinase from thermal inactivation. d -alanine and beta-alanine were not inhibitory to liver pyruvate kinase and neither these amino acids nor l -alanine inhibited muscle pyruvate kinase. 9. (9)| l -alanine exerted no effect on hepatic glucokinase, hexokinase, glucose 6-phosphatase or fructose 1,6-diphosphatase when added to the incubation mixture, or with preincubation, or at different levels of the respective substrates. 10. (10)|The factors regulating activity and biosynthesis of liver pyruvate kinase were reviewed and discussed.