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 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 Secondary-site binding of Glu-plasmin, Lys-plasmin and miniplasmin to fibrin

1981 ◽  
Vol 197 (3) ◽  
pp. 619-628 ◽  
Author(s):  
Elisabeth Suenson ◽  
Sixtus Thorsen
Keyword(s):  
Tranexamic Acid ◽  
Binding Site ◽  
Active Site ◽  
Marked Change ◽  
Secondary Site ◽  
Minor Importance ◽  
Kunitz Inhibitor ◽  
Pancreatic Trypsin Inhibitor ◽  
Human Plasminogen ◽  
Plasmin Inhibitor

Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1–790)]. This binding is decreased by α2-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, α2-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu1–Lys76, Glu1–Arg67or Glu1–Lys77)display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, α2-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys.170, 300–305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of α2-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu1–Val441) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by α2-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than α2-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.

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 Inactivation of skeletal-muscle UDP-glucose pyrophosphorylase by reaction with carboxylate-directed reagents

1989 ◽  
Vol 264 (3) ◽  
pp. 799-804 ◽  
Author(s):  
M Signorini ◽  
C Ferrari ◽  
E Mariotti ◽  
F Dallocchio ◽  
C M Bergamini
Keyword(s):  
Skeletal Muscle ◽  
Kinetic Analysis ◽  
Binding Site ◽  
Active Site ◽  
Enzyme Inactivation ◽  
Carboxylate Group ◽  
Hydrophobic Domain ◽  
Selective Protection ◽  
Carboxylate Groups ◽  
Affinity Labelling

Skeletal-muscle UDP-glucose pyrophosphorylase is inactivated by reaction with 2-ethoxy-N-(ethoxy-carbonyl)-1,2-dihydroquinoline (EEDQ) and 1-(3-dimethylaminopropyl-3-ethylcarbodi-imide (EDAC), two reagents specific for carboxylate groups. The former reagent is a more effective inactivator than EDAC. Although no evidence of reversible enzyme-reagent complexes of the affinity-labelling type was obtained by kinetic analysis of the inactivation, the selective protection of UDP-glucose pyrophosphorylase activity against inactivation by EEDQ in the presence of uridine substrates is indicative of an active-site-directed effect. The results are consistent with the hypothesis that EEDQ modifies a single carboxylate group located in a hydrophobic domain close to the substrate-binding site, leading to enzyme inactivation. In contrast, the reaction between UDP-glucose pyrophosphorylase and EDAC appears to involve a different region of the enzyme.

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

1998 ◽  
Vol 17 (7) ◽  
pp. 699-712 ◽  
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

scholarly journals Mammalian Spermidine Synthase—Identification of Cysteine Residues and Investigation of the Putrescine Binding Site—

2004 ◽  
Vol 27 (9) ◽  
pp. 1327-1332 ◽  
Author(s):  
Hitomi Goda ◽  
Toshiko Watanabe ◽  
Noboru Takeda ◽  
Masaki Kobayashi ◽  
Makiko Wada ◽  
...  
Keyword(s):  
Binding Site ◽  
Cysteine Residues ◽  
Spermidine Synthase
Sign in / Sign up

Export Citation Format

Share Document