Structure-Activity Studies on Mammalian Adenosine Kinase

Maj, Mary C.; Singh, Bhag; Gupta, Radhey S.

doi:10.1006/bbrc.2000.3307

Cited by 19 publications

(22 citation statements)

References 34 publications

(49 reference statements)

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“…The catalytic activity of the D299A mutant was completely knocked out, indicating that this residue is absolutely indispensable for catalytic function of LdAdK. Our result reaffirmed the importance of this highly conserved residue that had been suggested by other investigators [20,22,23].…”

Section: Kinetic Properties Of Different Mutantssupporting

confidence: 88%

“…A preliminary analysis of the hamster AdK by Maj et al [23] provided more direct evidence for the involvement of an aspartate residue in proper functioning of the enzyme. Our model of LdAdK also showed that Asp-299 (corresponding to Asp-300 of human and Asp-318 of T. gondii AdK) is ideally positioned for interaction with the 5 hydroxy group of ribose.…”

Section: Discussionmentioning

confidence: 99%

“…Among them, the carboxy group of a highly conserved aspartate residue (Asp-318 for T. gondii AdK, and Asp-300 for human AdK) has been shown to form a hydrogen bond with the ribosyl O5 hydroxy group of Ado and is predicted to have important role in catalysis [20,22]. Despite the predictions made from the structural analysis, apart from a preliminary report [23], actual biochemical evidence explaining the catalytic mechanism and substrate-binding characteristics still remains to be validated. Leishmania donovani, a purine auxotrophic protozoan parasite that has a dimorphic life cycle, is the causative agent of visceral leishmaniasis in humans.…”

Section: Introductionmentioning

confidence: 98%

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Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani

Datta

Das

Sen

et al. 2005

Biochemical Journal

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Leishmania donovani adenosine kinase (LdAdK) plays a pivotal role in scavenging of purines from the host. Exploiting interspecies homology and structural co-ordinates of the enzyme from other sources, we generated a model of LdAdK that led us to target several amino acid residues (namely Gly-62, Arg-69, Arg-131 and Asp-299). Replacement of Gly-62 with aspartate caused a drastic reduction in catalytic activity, with decreased affinity for either substrate. Asp-299 was found to be catalytically indispensable. Mutation of either Arg-131 or Arg-69 caused a significant reduction in k cat . R69A (Arg-69 → Ala) and R131A mutants exhibited unaltered K m for either substrate, whereas ATP K m for R69K increased 6-fold. Importance of both of the arginine residues was reaffirmed by the R69K/R131A double mutant, which exhibited approx. 0.5 % residual activity with a large increase in ATP K m . Phenylglyoxal, which inhibits the wild-type enzyme, also inactivated the arginine mutants to different extents. Adenosine protected both of the Arg-69 mutants, but not the R131A variant, from inactivation. Binding experiments revealed that the AMPbinding property of R69K or R69A and D299A mutants remained largely unaltered, but R131A and R69K/R131A mutants lost their AMP binding ability significantly. The G62D mutant did not bind AMP at all. Free energy calculations indicated that Arg-69 and Arg-131 are functionally independent. Thus, apart from the mandatory requirement of flexibility around the diglycyl (Gly-61-Gly-62) motif, our results identified Asp-299 and Arg-131 as key catalytic residues, with the former functioning as the proton abstractor from the 5 -OH of adenosine, while the latter acts as a bidentate electrophile to stabilize the negative charge on the leaving group during the phosphate transfer. Moreover, the positive charge distribution of Arg-69 probably helps in maintaining the flexibility of the α-3 helix needed for proper domain movement. These findings provide the first comprehensive biochemical evidence implicating the mechanistic roles of the functionally important residues of this chemotherapeutically exploitable enzyme.

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Section: Kinetic Properties Of Different Mutantssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

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Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani

Datta

Das

Sen

et al. 2005

Biochemical Journal

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“…In addition, mutation of this aspartic residue (D316) in mammalian adenosine kinase into either asparagine or glutamic acid, leads to a complete loss of activity (27). Similar results were obtained by mutation of the corresponding residue in the adenosine kinase from Leishmania donovani (D299) (28) and Homo sapiens (D300) (23).…”

Section: Catalytic Mechanismsupporting

confidence: 61%

The ADP‐dependent sugar kinase family: Kinetic and evolutionary aspects

Guixé

Merino

2009

IUBMB Life

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SummarySome archaea of the Euryarchaeota present a unique version of the Embden-Meyerhof pathway where glucose and fructose-6-phosphate are phoshporylated using ADP instead of ATP as the phosphoryl donor. These are the only ADP-dependent kinases known to date. Although initially they were believed to represent a new protein family, they can be classified as members of the ribokinase superfamily, which also include several ATP-dependent kinases. As they were first identified in members of the thermococcales it was proposed that the presence of these ADP-dependent kinases is an adaptation to high temperatures. Later, homologs of these enzymes were identified in the genomes of mesophilic and thermophilic methanogenic archaea and even in the genomes of higher eukaryotes, suggesting that the presence of these proteins is not related to the hyperthermophilic life. The ADP-dependent kinases are very restrictive to their ligands being unable to use triphosphorylated nucleotides such as ATP. However, it has been shown that they can bind ATP by competition kinetic experiments. The hyperthermophilic methanogenic archaeon Methanocaldococcus jannaschii has a homolog of these genes, which can phosphorylate glucose and fructose-6-phosphate. For this reason, it was proposed as an ancestral form for the family. However, recent studies have shown that the ancestral activity in the group is glucokinase, and a combination of gene duplication and lateral gene transfer could have originated the two paralogs in this member of the Euryarchaeota. Interestingly, based on structural comparisons made within the superfamily it has been suggested that the ADP-dependent kinases are the newest in the group. In several members of the superfamily, the presence of divalent metal cations has been shown to be crucial for catalysis, so its role in the ADP-dependent family was investigated through molecular dynamics. The simulation shows that, in fact, the metal coordinates the catalytic ensemble and interacts with crucial residues for catalysis. Keywords ADP-dependent kinase family; ribokinase superfamily; modified Embden-Meyerhof pathway; glucokinase; phosphofructokinase.Abbreviations ADP-GK, ADP-dependent glucokinase; ADP-PFK, ADP-dependent phosphofructokinase; ThzK, 4-Methyl-5-b-hydroxyethylthiazole kinase; HmppK, 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase.

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“…To exhibit antiviral activity, compound 1 should be converted to the triphosphate form. Adenosine kinase is postulated to be the first enzyme in this intracellular sequence, converting compound 1 to a monophosphate form (18). To test this hypothesis, we cloned and expressed the human adenosine kinase using an E. coli system.…”

Section: Chemistrymentioning

confidence: 99%

Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside

Chen

Yin

Duraiswamy

et al. 2010

Antimicrob Agents Chemother

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We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxymethyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC 50 ) and cytotoxic concentration (CC 50 ) values of 0.7 M and >100 M, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14 C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis.The family Flaviviridae consists of three genera, Flavivirus, Pestivirus, and Hepacivirus. Many viruses from the genus Flavivirus are arthropod-borne and cause significant human diseases. The four serotypes of dengue virus (DENV) alone pose a health threat to 2.5 billion people worldwide, leading to 50 to 100 million human infections and 500,000 cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) each year (11). Besides DENV, other flaviviruses, such as West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV), also cause frequent outbreaks throughout the world. No clinically approved antiviral therapy is currently available for treatment of flavivirus infections. Human vaccines are available only for YFV, JEV, and TBEV. The development of a successful DENV vaccine has been challenging due to the facts that (i) the vaccine should simultaneously induce a long-lasting protection against all four DENV serotypes and (ii) an incompletely immunized individual may be sensitized to the lifethreatening DHF or DSS. The challenge associated with the development of a vaccine underlines the importance of antiviral development for DENV and other flaviviruses.The flavivirus genome is a single-strand RNA of plus-sense polarity. The single open reading frame from the 11-kb viral genome encodes three structural proteins (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Nonstructural proteins are responsible for viral replication. Of those, NS3 and NS5 have enzymatic activities. The N-terminal domain of NS3 (with NS2B as a cofactor) functions as a serine protease, and the C-terminal domain acts as an RNA helicase, an RNA triphosphatase, and an NTPase (10,34,35). The N-...

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Structure-Activity Studies on Mammalian Adenosine Kinase

Cited by 19 publications

References 34 publications

Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani

Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani

The ADP‐dependent sugar kinase family: Kinetic and evolutionary aspects

Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside

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