Crystal Structures of Adenylosuccinate Synthetase fromEscherichia coliComplexed with GDP, IMP Hadacidin, NO3–, and Mg2 +

Poland, Bradley W.; Fromm, Herbert J.; Honzatko, Richard B.

doi:10.1006/jmbi.1996.0693

Cited by 48 publications

(121 citation statements)

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“…Synergism in the binding of IMP and GTP is suggested by studies of Wang et al (21). Furthermore, Mg 2ϩ binds to guanine nucleotides and putatively to the ␣-carboxylate of aspartate (5). Thus, the observed 6-fold increase in the K m for aspartate may be due entirely to the 6-fold increase in the K m for GTP and, presumably, bound Mg 2ϩ .…”

Section: Resultsmentioning

confidence: 90%

“…Two Mg 2ϩ ions are involved in the reaction mechanism (4). One Mg 2ϩ is in the active site, associated with the phosphate moiety of the guanine nucleotide and the N-formyl group of hadacidin, an inactive analog of aspartate (5). However, crystallographic investigations have yet to reveal the location of the second Mg 2ϩ .…”

mentioning

confidence: 99%

“…On the basis of preliminary crystal structures of ligated AMPSase, which are now complete (5,6), several residues are in positions of putative significance to catalytic function, ligand binding, and/or structural organization of the active site. Asp 13 of AMPSase hydrogen bonds with N-1 of IMP and approaches the sixth coordination site of a pentavalently coordinated Mg 2ϩ .…”

mentioning

confidence: 99%

“…Asp 13 of AMPSase hydrogen bonds with N-1 of IMP and approaches the sixth coordination site of a pentavalently coordinated Mg 2ϩ . Asp 13 is putatively a catalytic base in the abstraction of the proton from N-1 of IMP (5). Glu 14 hydrogen bonds to the backbone amides 10 and 12 of the P-loop (residues 8 -17 of AMPSase) and to NZ of Lys 16 .…”

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confidence: 99%

“…Lys 16 of AMPSase corresponds to the essential lysine of the consensus P-loop sequence GXXXXGK (7,8). Lys 16 interacts with the ␤-phosphate of GDP and/or anions (nitrate and phosphate) bound to the ␥-phosphoryl site (5,6). Lys 16 putatively stabilizes the pentavalent transition state of the ␥-phosphoryl group during the phosphotransfer reaction.…”

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confidence: 99%

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Residues Essential for Catalysis and Stability of the Active Site of Escherichia coli Adenylosuccinate Synthetase as Revealed by Directed Mutation and Kinetics

Kang

Sun

Poland

et al. 1997

Journal of Biological Chemistry

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Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp 13 , Glu 14 , Lys 16 , His 41 , and Arg 131 , to the catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no measurable activity. Mutants E14A and H41N exhibit 1% of the activity of the wild-type enzyme and 2-7-fold increases in the K m of substrates. The mutant K16Q has 34% of the activity of wild-type enzyme and K m values for substrates virtually unchanged from those of the wild-type system. Mutation of Arg 131 to leucine caused only a 4-fold increase in the K m for aspartate relative to the wild-type enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on k cat are consistent with the putative roles assigned to Asp 13 (catalytic base), His 41 (catalytic acid), and Glu 14 (structural organization of the active site). The modest effect of the R131L mutation on the binding of aspartate is also in harmony with recent crystallographic investigations, which suggests that Arg 131 stabilizes the conformation of the loop that binds the ␤-carboxylate of aspartate. The modest effect of the K16Q mutation, however, contrasts with significant changes brought about by the mutation of the corresponding lysines in the P-loop of other GTPand ATP-binding proteins. Crystallographic structures place Lys 16 in a position of direct interaction with the ␥-phosphate of GTP. Furthermore, lysine is present at corresponding positions in all known sequences of adenylosuccinate synthetase. We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys 16 may stabilize the enzyme structurally. In addition, the modest loss of catalytic activity of the K16Q mutant may confer such a selective disadvantage to E. coli that this seemingly innocuous mutation is not tolerated in nature.Adenylosuccinate synthetase (AMPSase) 1 (see Ref. 1 for review) catalyzes the following reversible reaction in the presence of Mg 2ϩ ions: GTP ϩ IMP ϩ aspartate i GDP ϩ adenylosuccinate ϩ phosphate (P i ). This reaction is the first committed step in the formation of AMP from IMP on the pathway for de novo purine nucleotide biosynthesis and is an integral part of the purine nucleotide cycle in muscle (2). The reaction mechanism of AMPSase centers on 6-phosphoryl-IMP, formed putatively by the nucleophilic attack of the 6-oxyanion of IMP on the ␥-phosphate of GTP. A second nucleophilic substitution reaction by the amino group of aspartate on the C-6 of 6-phosphoryl-IMP yields adenylosuccinate and P i (3). Two Mg 2ϩ ions are involved in the reaction mechanism (4). One Mg 2ϩ is in the active site, associated with the phosphate moiety of the guanine nucleotide and the N-formyl group of hadacidin, an inactive analog of aspartate (5). However, crystallographic investigations have yet to reveal the location of the second Mg 2ϩ

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Section: Resultsmentioning

confidence: 90%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Residues Essential for Catalysis and Stability of the Active Site of Escherichia coli Adenylosuccinate Synthetase as Revealed by Directed Mutation and Kinetics

Kang

Sun

Poland

et al. 1997

Journal of Biological Chemistry

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Leishmania donovani adenylosuccinate synthetase requires IMP for dimerization and organization of the active site

Mochi,

Jani,

Shah

et al. 2024

FEBS Letters

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Adenylosuccinate synthetase (AdSS), which catalyses the GTP‐dependent conversion of inosine monophosphate (IMP) and aspartic acid to succinyl‐AMP, plays a major role in purine biosynthesis. In some bacterial AdSS, it is implicated that IMP binding is important to organize the active site, but in certain plant AdSS, GTP performs this role. Here, we report that in Leishmania donovani AdSS, IMP binding favoured dimerization, induced greater conformational change and improved the protein stability more than GTP binding. IMP binding, which resulted in a network of hydrogen bonds, stabilized the conformation of active site loops and brought the switch loop to a closed conformation, which then facilitated GTP binding. Our results provide a basis for designing better inhibitors of leishmanial AdSS.

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Adenylosuccinate Syntheatase: Recent Developments

Honzatko

Stayton

Formm

1999

Advances in Enzymology - And Related Areas of Molecular Biology

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Crystal Structures of Adenylosuccinate Synthetase fromEscherichia coliComplexed with GDP, IMP Hadacidin, NO3–, and Mg2 +

Cited by 48 publications

References 47 publications

Residues Essential for Catalysis and Stability of the Active Site of Escherichia coli Adenylosuccinate Synthetase as Revealed by Directed Mutation and Kinetics

Residues Essential for Catalysis and Stability of the Active Site of Escherichia coli Adenylosuccinate Synthetase as Revealed by Directed Mutation and Kinetics

Leishmania donovani adenylosuccinate synthetase requires IMP for dimerization and organization of the active site

Adenylosuccinate Syntheatase: Recent Developments

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