Glutamine hydrolysis

Bridging the 40-A gap between glutamine hydrolysis and GIn-tRNA " synthesis 396... 

Figure 25.3. Structure of Carbamoyl Phosphate Synthetase. This enzyme consists of two chains. The smaller chain (yellow) contains a site for glutamine hydrolysis to generate ammonia. The larger chain includes two ATP-grasp domains (blue and red). In one ATP-grasp domain (blue), bicarbonate is phosphorylated to carboxyphosphate, which then reacts with ammonia to generate carbamic acid. In the other ATP-grasp domain, the carbamic acid is phosphorylated to produce carbamoyl phosphate.

These values are calculated from the stoichiometry of glutamate to 5 -phosphorlbosylamlhe, ahd therefore represent the fractloh of ammonia released by glutamine hydrolysis that Is used In the synthase reaction. All values for the mutaht ehzymes are normalized to that of WT GPATase (0.90). 

The mechanism of glutamine hydrolysis is reminiscent of the thiol proteases. A thioester intermediate (Figure 4) is formed with the release of ammonia and then hydrolyzed in the subsequent step regenerating the active site for another round of catalysis (40-43). The formation and breakdown of the thioester involves the participation of a catalytic triad. 

Figure 4. Mechanism of Glutamine Hydrolysis. The y-glutamyl thioester is formed (ks) with the release ofNHs and hydrolyzed (k4) in a subsequent step.

Table II. Kinetics of Glutamine Hydrolysis of tiie Mammalian GLN- E, coli...

The amino half of the GLN domain was also expressed as a soluble compactly folded protein in E. coli. As expected, the 19 kDa subdomain had no catalytic activity but could form a stable complex with the E. coli CPSase synthetase subunit (Figure 5), indicating that it is crucial for the formation of the GLN CPS complex. This region of the molecule, now designated the attenuation subdomain, suppresses the high intrinsic catalytic activity of the catalytic subdomain. Excision of the attenuation subdomain from GLN results in a 14,000-fold increase in the apparent second order rate for glutamine hydrolysis. 

AG° for this reaction is -14-3 kJ moP, i.e. the equilibrium position of the reaction lies far to the right. From the previous discussion, the value of AG for this reaction will be negative at physiological concentrations of glutamine and glutamate and the reaction will proceed in the direction of glutamine hydrolysis. 

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