Conformation of neolignans that bind to the arginine residue in adenosine-kinase from Leishmania donovani

1999 ◽  
Vol 464 (1-3) ◽  
pp. 281-287 ◽  
Author(s):  
Maria Cristina Andreazza Costa ◽  
Lauro Euclides Soares Barata ◽  
Yuji Takahata
Keyword(s):  
Leishmania Donovani ◽  
Adenosine Kinase ◽  
Arginine Residue

scholarly journals Probing the function(s) of active-site arginine residue in Leishmania donovani adenosine kinase

1994 ◽  
Vol 298 (2) ◽  
pp. 295-301 ◽  
Author(s):  
M Ghosh ◽  
A K Datta
Keyword(s):  
Active Site ◽  
Leishmania Donovani ◽  
Order Rate Constant ◽  
Adenosine Kinase ◽  
Arginine Residue ◽  
Affinity Column ◽  
Protein Fluorescence ◽  
Original Activity ◽  
Modified Enzyme ◽  
Sepharose 4B

The presence of arginine at the active site of Leishmania donovani adenosine kinase was studied by chemical modification, followed by the characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), butane-2,3-dione and cyclohexane-1,2-dione all irreversibly inactivated the enzyme. In contrast, adenosine kinase from hamster liver was insensitive to these reagents. The inactivation of the enzyme by PGO followed pseudo-first-order kinetics, with a second-order rate constant of 39.2 min-1.M-1. Correlation between the stoichiometry of PGO modification and extent of inactivation indicated that modification of a single residue per molecule suffices for the loss of activity. Reactivity of the essential arginine residue towards PGO was affected by the presence of adenosine (Ado) and other competing alternative substrates, consistent with an arginine residue located proximal to the Ado-binding site. The enzyme showed an intrinsic fluorescence with an emission maximum at 340 nm when excited at 295 nm. The protein fluorescence was partially quenched on addition of Ado. PGO modification also led to significant quenching of the fluorescence. However, the fluorescence of the Ado-protected enzyme, which displayed 82% of the original activity after PGO treatment, was retained. The kinetic analyses of the partially modified enzyme showed an increase in the Km for Ado from 14 to 55 microM. Furthermore, the inability of the modified enzyme to bind to 5′-AMP-Sepharose 4B affinity column provided additional evidence that modification is attended by decrease in affinity of the enzyme for Ado. The results are consistent with the interpretation that modification of the active-site arginine residue affects activity by interfering with the binding of the substrate to the active site.

scholarly journals Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif

1999 ◽  
Vol 339 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Krishna Murari SINHA ◽  
Mallika GHOSH ◽  
Ishita DAS ◽  
Alok K. DATTA
Keyword(s):  
Leishmania Donovani ◽  
Tryptophan Fluorescence ◽  
Biochemical Properties ◽  
Adenosine Kinase ◽  
Cloning And Expression ◽  
Translation Initiation Site ◽  
Gene Transcript ◽  
Protein Alignment ◽  
Significant Similarity ◽  
Calculated Molecular Mass

The unique catalytic characteristics of adenosine kinase (Adk) and its stage-specific differential activity pattern have made this enzyme a prospective target for chemotherapeutic manipulation in the purine-auxotrophic parasitic protozoan Leishmania donovani. However, nothing is known about the structure of the parasite Adk. We report here the cloning of its gene and the characterization of the gene product. The encoded protein, consisting of 345 amino acid residues with a calculated molecular mass of 37173 Da, shares limited but significant similarity with sugar kinases and inosine-guanosine kinase of microbial origin, supporting the notion that these enzymes might have the same ancestral origin. The identity of the parasite enzyme with the corresponding enzyme from two other sources so far described was only 40%. Furthermore, 5ʹ RNA mapping studies indicated that the Adk gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon (spliced leader) occurring at nt -160 from the predicted translation initiation site. The biochemical properties of the recombinant enzyme were similar to those of the enzyme isolated from leishmanial cells. The intrinsic tryptophan fluorescence of the enzyme was substrate-sensitive. On the basis of a multiple protein-alignment sequence comparison and ATP-induced fluorescence quenching in the presence or the absence of KI and acrylamide, the docking site for ATP has been provisionally identified and shown to have marked divergence from the consensus P-loop motif reported for ATP- or GTP-binding proteins from other sources.

scholarly journals Two conformationally vicinal thiols at the active site of Leishmania donovani adenosine kinase

1996 ◽  
Vol 316 (2) ◽  
pp. 439-445 ◽  
Author(s):  
Tapan K. BAGUI ◽  
Mallika GHOSH ◽  
Alok K. DATTA
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Leishmania Donovani ◽  
Marked Change ◽  
Adenosine Kinase ◽  
Inactivation Kinetics ◽  
Cysteine Residues ◽  
Schematic Model ◽  
Hydrophobic Domain ◽  
Modified Enzyme

Inactivation of adenosine kinase (Adk) from Leishmania donovani correlates with the modification of two conformationally vicinal cysteine residues. In contrast, Adk from hamster liver, despite being sensitive to monothiol-blocking reagents, was insensitive to dithiol modifiers. Inactivation kinetics and substrate-protection studies along with double-modification experiments successively with N-ethylmaleimide in the presence of Ado and sodium m-arsenite–2,3-dimercaptopropanol or diazenedicarboxylic acid bis-N,N´-dimethylamide supported assignment of the two thiols at the Ado-binding site. Cystine bridge formation impaired the ability of the modified enzyme to bind to the substrate. Tryptophan fluorescence of the enzyme was quenched after modification by dithiol-blocking reagents with concomitant loss of activity. However, treatment of the enzyme with methylmethanethiosulphonate (MMTS) led to complete inactivation without a marked change in protein fluorescence. Ado protected both fluorescence and catalytic activity against inactivation by both MMTS and dithiol-blocking reagents. Stern–Volmer quenching analysis of the native and Ado-complexed enzyme suggested that, of the four tryptophan residues, at least one is located at or near the active site. Furthermore quenching constants of native, Ado-complexed and dithiol-modified enzyme in the presence of either acrylamide or KI indicated spatial proximity of tryptophan and two cysteine residues within the hydrophobic domain of the Ado-binding site. Taken together the results suggest important function(s) for the cysteine residue(s). A schematic model is proposed to explain the inactivation of the enzyme by both monothiol- and dithiol-blocking reagents.

scholarly journals Computational Elucidation of Structural Basis for Ligand Binding withLeishmania donovaniAdenosine Kinase

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Rajiv K. Kar ◽  
Md. Yousuf Ansari ◽  
Priyanka Suryadevara ◽  
Bikash R. Sahoo ◽  
Ganesh C. Sahoo ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Active Site ◽  
Protein Complex ◽  
Leishmania Donovani ◽  
Homology Model ◽  
Adenosine Kinase ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Active Site Residues ◽  
Drug Candidates

Enzyme adenosine kinase is responsible for phosphorylation of adenosine to AMP and is crucial for parasites which are purine auxotrophs. The present study describes development of robust homology model ofLeishmania donovaniadenosine kinase to forecast interaction phenomenon with inhibitory molecules using structure-based drug designing strategy. Docking calculation using reported organic small molecules and natural products revealed key active site residues such as Arg131 and Asp16 for ligand binding, which is consistent with previous studies. Molecular dynamics simulation of ligand protein complex revealed the importance of hydrogen bonding with active site residues and solvent molecules, which may be crucial for successful development of drug candidates. Precise role of Phe168 residue in the active site was elucidated in this report that provided stability to ligand-protein complex via aromatic-πcontacts. Overall, the present study is believed to provide valuable information to design a new compound with improved activity for antileishmanial therapeutics development.

scholarly journals Homology-model-guided site-specific mutagenesis reveals the mechanisms of substrate binding and product-regulation of adenosine kinase from Leishmania donovani

2006 ◽  
Vol 394 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Rupak Datta ◽  
Ishita Das ◽  
Banibrata Sen ◽  
Anutosh Chakraborty ◽  
Subrata Adak ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Enzyme Regulation ◽  
Fold Increase ◽  
Homology Model ◽  
Binding Pocket ◽  
Adenosine Kinase ◽  
Binding Studies ◽  
Site Specific ◽  
Product Regulation ◽  
Amp Inhibition

Despite designating catalytic roles of Asp299 and Arg131 during the transfer of γ-phosphate from ATP to Ado (adenosine) [R. Datta, Das, Sen, Chakraborty, Adak, Mandal and A. K. Datta (2005) Biochem. J. 387, 591–600], the mechanisms that determine binding of substrate and cause product inhibition of adenosine kinase from Leishmania donovani remained unclear. In the present study, employing homology-model-guided site-specific protein mutagenesis, we show that Asp16 is indispensable, since its replacement with either valine or arginine resulted in a >200-fold increase in Km (Ado) with a 1000-fold decrease in kcat/Km, implying its critical importance in Ado binding. Even glutamate replacement was not tolerated, indicating the essentiality of Asp16 in the maintenance of steric complementarity of the binding pocket. Use of 2′or 3′-deoxygenated Ado as substrates indicated that, although both the hydroxy groups play important roles in the formation of the enzyme–Ado complex, the binding energy (ΔΔGB) contribution of the former was greater than the latter, suggesting possible formation of a bidentate hydrogen bond between Asp16 and the adenosyl ribose. Interestingly, AMP-inhibition and AMP-binding studies revealed that, unlike the R131A mutant, which showed abrogated AMP-binding and insensitivity towards AMP inhibition despite its unaltered Km (Ado), all the Asp16 mutants bound AMP efficiently and displayed AMP-sensitive catalytic activity, suggesting disparate mechanisms of binding of Ado and AMP. Molecular docking revealed that, although both Ado and AMP apparently occupied the same binding pocket, Ado binds in a manner that is subtly different from AMP binding, which relies heavily on hydrogen-bonding with Arg131 and thus creates an appropriate environment for competition with Ado. Hence, besides its role in catalysis, an additional novel function of the Arg131 residue as an effector of product-mediated enzyme regulation is proposed.

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