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Inhibition by glutamate of phosphate-dependent glutaminase of rat kidney
Abstract:
A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.
Insights
Phosphate activates membrane-bound glutaminase, altering its conformation and substrate affinity. This phosphate-dependent regulation affects how glutamine and glutamate bind to the enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Mitochondrial Function
Background:
- Phosphate-dependent glutaminase is an enzyme crucial for cellular metabolism.
- Understanding its regulation is key to comprehending metabolic pathways.
- Previous studies indicated phosphate influences solubilized glutaminase.
Purpose of the Study:
- To characterize the phosphate-dependent regulation of membrane-associated glutaminase.
- To investigate the conformational changes induced by phosphate.
- To determine the kinetic parameters of glutamate inhibition.
Main Methods:
- Purification of membrane-associated phosphate-dependent glutaminase from mitochondria.
- Enzyme kinetics studies using radioactive assays.
- Analysis of enzyme inactivation rates with affinity labels.
- Determination of Michaelis constant (Km) and inhibition constants (Ki).
Main Results:
- Phosphate sigmoidaly activates membrane-bound glutaminase.
- Phosphate induces similar conformational changes in both membrane-bound and solubilized forms.
- Glutamine Km decreases significantly with increasing phosphate concentration.
- Glutamate acts as a competitive inhibitor, with its Ki increasing with phosphate levels.
Conclusions:
- Membrane-associated glutaminase undergoes phosphate-induced conformational changes.
- Phosphate concentration modulates the enzyme's affinity for glutamine and glutamate.
- Enzyme specificity is regulated by phosphate availability at the active site.