Catalysis Uncoupling in a Glutamine Amidotransferase Bienzyme by Unblocking the Glutaminase Active Site

Abstract

Nitrogen is incorporated into various metabolites by multifunctional glutamine amidotransferases via reactive ammonia generated by glutaminase hydrolysis of glutamine. Although this process is generally tightly regulated by subsequent synthase activity, little is known about how the glutaminase is inhibited in the absence of an activating signal. Here, we use imidazoleglycerolphosphate synthase as a model to investigate the mechanism of glutaminase regulation. A structure of the bienzyme-glutamine complex reveals that the glutaminase active site is in a catalysis-competent conformation but the ammonia pathway toward the synthase active site is blocked. Mutation of two residues blocking the pathway leads to a complete uncoupling of the two reactions and to a 2800-fold amplification of glutaminase activity. Our data advance the understanding of coupling enzymatic activities in glutamine amidotransferases and raise hypotheses of the underlying molecular mechanism.

Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

“…Interestingly, despite very similar chemical shift behavior between the two, K19A IGPS is approximately threefold more active than D98A, consistent with earlier work by Davisson, Wilmanns, and coworkers (30,31) on the K19A and D98A enzymes, respectively. V12A is the only mutation that retains WT-like glutaminase activity with a k cat value that is 50% that of WT IGPS, resulting in a similar reduction in catalytic efficiency.…”

Dissecting Dynamic Allosteric Pathways Using Chemically Related Small-Molecule Activators

“…In support of this, mutagenesis of residues f D98, f K99, h Y138 and h K181, which are adjacent to these residues cause a varying degree of allosteric defects in IGPS, presumably through disruption of the interfacial salt bridges proposed to regulate NH 3 transport to HisF. (List et al, 2012) Further, the significant number of ligand-dependent differences in this synergy, especially those found in comparison to the PRFAR-bound ternary complex, indicates there are likely multiple allosteric networks within IGPS. Thus, the best allosteric activator does not cause large chemical shifts changes in the most residues in comparison to other ligands; but PRFAR binding does result in the largest number of residues with synergistic chemical shift changes.…”

“…Crystallographic(Chaudhuri et al, 2001; Chaudhuri et al, 2003; Douangamath et al, 2002), NMR(Lipchock et al, 2010), and molecular dynamics(Manley et al, 2013; Rivalta et al, 2012) studies suggest a rotation of the HisH active site h Pro49- h Gly50- h Val51- h Gly52 (PGVG) loop, which lies adjacent to the HisH catalytic triad, is required for proper formation of the oxyanion hole during catalysis, although double-mutant studies suggest additional factors are essential for HisH catalysis. (List et al, 2012) To examine long-range perturbations at the glutaminase site caused by effector binding, we monitored the amide backbone chemical shifts of the h G50 and h G52 resonances of the PGVG loop during allosteric ligand titration into 2 H, 15 N-labeled HisH-IGPS (Figure 4). …”

Glutaminase 1 is a potential biomarker for chronic post-surgical pain in the rat dorsal spinal cord using differential proteomics

“…Glutamic acid (Glu) in vivo is produced from glutamine deamination (Erickson and Cerione 2010;List et al 2012;Marquez et al 2013). Glu is the most important excitatory amino acid in the excitatory neurotransmitter system, which plays a key role in learning, memory and central pain sensory conduction (de la Rosa et al 2009;Marquez et al 2006;Miller et al 2012).…”