Active site thiol(s) in Leishmania donovani adenosine kinase: comparison with hamster enzyme and evidence for the absence of regulatory adenosine binding site

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.

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

Biochemical Journal ◽

10.1042/bj2980295 ◽

1994 ◽

Vol 298 (2) ◽

pp. 295-301 ◽

Cited By ~ 13

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.

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Computational Elucidation of Structural Basis for Ligand Binding withLeishmania donovaniAdenosine Kinase

BioMed Research International ◽

10.1155/2013/609289 ◽

2013 ◽

Vol 2013 ◽

pp. 1-14 ◽

Cited By ~ 25

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.

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Site-directed mutagenesis of histidine 93, aspartic acid 180, glutamic acid 205, histidine 290, and aspartic acid 291 at the active site and tryptophan 279 at the raw starch binding site in barley alpha-amylase 1.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)41554-2 ◽

1993 ◽

Vol 268 (30) ◽

pp. 22480-22484

Author(s):

M Søgaard ◽

A Kadziola ◽

R Haser ◽

B Svensson

Keyword(s):

Glutamic Acid ◽

Binding Site ◽

Aspartic Acid ◽

Active Site ◽

Site Directed Mutagenesis ◽

Alpha Amylase ◽

Directed Mutagenesis ◽

Raw Starch ◽

Raw Starch Binding

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The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3)

Journal of Protein Chemistry ◽

10.1007/bf02780973 ◽

1998 ◽

Vol 17 (7) ◽

pp. 699-712 ◽

Cited By ~ 4

Author(s):

Dennis E. Epps ◽

Roger A. Poorman ◽

Gary L. Petzold ◽

Christopher W. Stuchly ◽

Alice L. Laborde ◽

...

Keyword(s):

Binding Site ◽

Fluorescent Probes ◽

Active Site

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Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

Journal of Structural Biology ◽

10.1016/j.jsb.2011.06.001 ◽

2011 ◽

Vol 176 (1) ◽

pp. 52-59 ◽

Cited By ~ 10

Author(s):

Vivian Cody ◽

Jim Pace ◽

Jennifer Piraino ◽

Sherry F. Queener

Keyword(s):

Binding Site ◽

Dihydrofolate Reductase ◽

Active Site ◽

Crystallographic Analysis ◽

Human Dihydrofolate Reductase

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Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

Biochemical Journal ◽

10.1042/bj20040697 ◽

2004 ◽

Vol 382 (2) ◽

pp. 751-757 ◽

Cited By ~ 23

Author(s):

Pakorn WINAYANUWATTIKUN ◽

Albert J. KETTERMAN

Keyword(s):

Binding Site ◽

Active Site ◽

Structural Integrity ◽

Catalytic Mechanism ◽

Tertiary Structure ◽

Site Directed Mutagenesis ◽

Active Site Residues ◽

Anopheles Dirus ◽

Steady State Kinetics ◽

Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

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Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17015813 ◽

2017 ◽

Vol 73 (12) ◽

pp. 651-656 ◽

Cited By ~ 1

Author(s):

Taichi Mizobuchi ◽

Risako Nonaka ◽

Motoki Yoshimura ◽

Katsumasa Abe ◽

Shouji Takahashi ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Active Site ◽

Metal Binding ◽

Bivalve Mollusc ◽

Active Site Residues ◽

X Ray ◽

Aspartate Racemase ◽

Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

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