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Mechanism of Fully Reversible, pH-Sensitive Inhibition of Human Glutamine Synthetase by Tyrosine Nitration

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posted on 2020-07-03, 18:03 authored by Benedikt Frieg, Boris Görg, Natalia Qvartskhava, Thomas Jeitner, Nadine Homeyer, Dieter Häussinger, Holger Gohlke
Glutamine synthetase (GS) catalyzes an ATP-dependent condensation of glutamate and ammonia to form glutamine. This reactionand therefore GSare indispensable for the hepatic nitrogen metabolism. Nitration of tyrosine 336 (Y336) inhibits human GS activity. GS nitration and the consequent loss of GS function are associated with a broad range of neurological diseases. The mechanism by which Y336 nitration inhibits GS, however, is not understood. Here, we show by means of unbiased MD simulations, binding, and configurational free energy computations that Y336 nitration hampers ATP binding but only in the deprotonated and negatively charged state of residue 336. By contrast, for the protonated and neutral state, our computations indicate an increased binding affinity for ATP. pKa computations of nitrated Y336 within GS predict a pKa of ∼5.3. Thus, at physiological pH, nitrated Y336 exists almost exclusively in the deprotonated and negatively charged state. In vitro experiments confirm these predictions, in that, the catalytic activity of nitrated GS is decreased at pH 7 and 6 but not at pH 4. These results indicate a novel, fully reversible, pH-sensitive mechanism for the regulation of GS activity by tyrosine nitration.

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