Phosphorylation Mechanism of N-Acetyl-l-glutamate Kinase, a QM/MM Study

Abstract

In microorganisms and plants, NAGK N-acetyl-L-glutamate kinase (NAGK) catalyses the second step in L-arginine synthesis, the phosphorylation of N-Acetyl-L-glutamate (NAG) to give N-acetyl-L-glutamate-5-phosphate (NAGP). NAGK is only present in microorganisms and plants but absent from mammals, which makes it an attractive target for antimicrobial or biocidal development. Understanding the substrate binding mode and reaction mechanism of NAGK is crucial for targeting the kinase to develop potential therapies. Here the substrate binding mode was studied by comparing the conformational change of NAGK in the presence and in the absence of the NAG substrate based on molecular dynamic simulations. We revealed that with substrate binding the catalytic site of the kinase involving three loops in NAGK exhibits a closed conformation, which is predominantly controlled by an interaction between Arg98 and the α-COOof NAG. Lys41 is found to guide phosphate transfer through the interactions with the β-,γ-, and γ-phosphate oxygen atoms of ATP surrounded by two highly conserved glycine residues (Gly44 and Gly76), while Arg98 helps to position the NAG substrate in the catalytic site, which facilitate the phosphate transfer. Furthermore, we elucidated phosphate transfer reaction mechanism using hybrid density functional theory-based QM/MM calculations (B97D/AMBER99) and found that the catalysis follows a dissociative mechanism.

Mechanism of the feedback-inhibition resistance in aspartate kinase of Corynebacterium pekinense: from experiment to MD simulations

“…The increased distance between the reactants was extremely detrimental for the catalytic activity of AK. 21 Superimposing the structures in the absence and presence of inhibitor lysine indicated that the conformations of the residues around the ATP binding site had no significant change (Fig. S2(B) † ).…”

“…To some extent, whether the catalytic reaction can occur or not can be simply judged by the distance between the phosphorus atom of the transferred phosphate (ATP) and the acceptor oxygen atom of the substrate Asp (d Pi-O ). [19][20][21] In this work, ATP, as a reactant, is directly involved in the catalysis of the AK enzyme, in which the gamma phosphate group transfers to the carboxyl group of the substrate Asp. The related reaction equation was shown in ref.…”

“…However, the d Pi-O curve for WT + Lys system uctuated drastically (4.01 AE 0.60Å) and ranged from 2.7 to 5.8 A across an interval of about 3Å, resulting in noncatalytic pose which would not facilitate phosphoryl transfer. [19][20][21] In contrast, the catalytic distances in two mutants were suitable for the a The bold indicates activation. reaction.…”

The priming phosphorylation of KaiC is activated by the release of its autokinase autoinhibition

“…It is important to discuss our observations with respect to previous QM/MM studies (see (29) and references are therein). QM/MM simulations have been used to investigate phosphoryl transfer reactions in several kinases, including cAMP-dependent protein kinase (30)(31)(32)(33)(34), cyclindependent kinase (35)(36)(37), and N-acetyl-L-glutamate kinase (38,39). In these examples, the activation energy for the phosphoryl transfer reaction ranges from 10 to 25 kcal mol -1 .…”

Quantum approach to the mechanism of monothiopyrophosphate isomerization

“…This contribution concentrates, however, on methodological aspects of calculations while employing only SMD/M06-2X/ 6311+G9d,p)//SMD/M06-2X/6-31+G(d) level of theory. Some other (non-exhaustive) recent examples of using different theory levels in modeling properties and reactivity of phosphoruscontaining molecules include DFTB3/3OB in structure determination [16,17], B3LYP & MP3 with 6-311G(d,p) basis set in conformational analysis [18], B3LYP/cc-pVTZ/TIP3P and B3LYP-D3/def2-SVP(D) in studies of anharmonicity of phosphate ions in water [19,20], M06-2X, M06L and PBE with 6-31+G(d,p) in lipophilicity calculations [21] ωB97/6-311+ G(3df,2p)//M06-2X/6-311+G(d,p) in investigations of the reaction with OH radical [22], MP3/6-311++G(2d,2p)//B3LYP/6 −31+G(d) in hydrolysis of pesticides [23], PM3/MM, BLYP/6-31G(d)/MM, B3LYP/6-31G*/MM and COSMO/B3LYP/6-31G(d) in hydrolysis of monoesters [24], B3LYP/6-311+ G(d,p) and M06-2X/6-311+G(d,p) in gas-phase decomposition [25], B3LYP/6-311+G(d,p) in metathiophosphate di-and polymerization [26], M06-2X/6-311+G(3df,2p) and B2PLYP/ 6311+G(3df,2p)//(IEFPCM) /M06-2X/6-311+G(d,p) in oxidation [27], PBE-D3/PAW in non-aqueous degradation [28], M06-2X/6-31G(d,p) and BLYP-D3/aug-cc-pVTZ in binding to an enzyme [29], and B97D/6-31+G(d)/AMBER [30], B3LYP/6-31G(d) [31] and PBE0-D3/def2-TZVP [32] in enzymatic catalysis.…”