Medhekar R


Full name : Medhekar Rohit

First name : Rohit

Mail : University of Iowa, Dept. of Chemistry, Iowa City, IA 52242

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Country : USA

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Phone : 319-335-1476

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References (6)

Title : Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases - Saxena_2003_Eur.J.Biochem_270_4447
Author(s) : Saxena A , Fedorko JM , Vinayaka CR , Medhekar R , Radic Z , Taylor P , Lockridge O , Doctor BP
Ref : European Journal of Biochemistry , 270 :4447 , 2003
Abstract : E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.
ESTHER : Saxena_2003_Eur.J.Biochem_270_4447
PubMedSearch : Saxena_2003_Eur.J.Biochem_270_4447
PubMedID: 14622273

Title : Importance of arginines 63 and 423 in modulating the bile salt-dependent and bile salt-independent hydrolytic activities of rat carboxyl ester lipase - Liang_2000_J.Biol.Chem_275_24040
Author(s) : Liang Y , Medhekar R , Brockman HL , Quinn DM , Hui DY
Ref : Journal of Biological Chemistry , 275 :24040 , 2000
Abstract : Previous studies using chemical modification approach have shown the importance of arginine residues in bile salt activation of carboxyl ester lipase (CEL) activity. However, the x-ray crystal structure of CEL failed to show the involvement of arginine residues in CEL-bile salt interaction. The current study used a site-specific mutagenesis approach to determine the role of arginine residues 63 and 423 in bile salt-dependent and bile salt-independent hydrolytic activities of rat CEL. Mutations of Arg(63) to Ala(63) (R63A) and Arg(423) to Gly(423) (R423G) resulted in enzymes with increased bile salt-independent hydrolytic activity against lysophosphatidylcholine, having 6.5- and 2-fold higher k(cat) values, respectively, in comparison to wild type CEL. In contrast, the R63A and R423A mutant enzymes displayed 5- and 11-fold decreases in k(cat), in comparison with wild type CEL, for bile salt-dependent cholesteryl ester hydrolysis. Although taurocholate induced similar changes in circular dichroism spectra for wild type, R63A, and R423G proteins, this bile salt was less efficient in protecting the mutant enzymes against thermal inactivation in comparison with control CEL. Lipid binding studies revealed less R63A and R423G mutant CEL were bound to 1,2-diolein monolayer at saturation compared with wild type CEL. These results, along with computer modeling of the CEL protein, indicated that Arg(63) and Arg(423) are not involved directly with monomeric bile salt binding. However, these residues participate in micellar bile salt modulation of CEL enzymatic activity through intramolecular hydrogen bonding with the C-terminal domain. These residues are also important, probably through similar intramolecular hydrogen bond formation, in stabilizing the enzyme in solution and at the lipid-water interface.
ESTHER : Liang_2000_J.Biol.Chem_275_24040
PubMedSearch : Liang_2000_J.Biol.Chem_275_24040
PubMedID: 10811659

Title : Theoretical and experimental investigations of electrostatic effects on acetylcholinesterase catalysis and inhibition - Malany_1999_Chem.Biol.Interact_119-120_99
Author(s) : Malany S , Baker NA , Verweyst M , Medhekar R , Quinn DM , Velan B , Kronman C , Shafferman A
Ref : Chemico-Biological Interactions , 119-120 :99 , 1999
Abstract : The role of electrostatics in the function of acetylcholinesterase (AChE) has been investigated by both theoretical and experimental approaches. Second-order rate constants (kE = k(cat)/Km) for acetylthiocholine (ATCh) turnover have been measured as a function of ionic strength of the reaction medium for wild-type and mutant AChEs. Also, binding and dissociation rate constants have been measured as a function of ionic strength for the respective charged and neutral transition state analog inhibitors m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) and m-(t-butyl)trifluoroacetophenone (TBTFA). Linear free-energy correlations between catalytic rate constants and inhibition constants indicate that kE for ATCh turnover is rate limited by terminal binding events. Comparison of binding rate constants for TMTFA and TBTFA attests to the sizable electrostatic discrimination of AChE. Free energy profiles for cationic ligand release from the active sites of wild-type and mutant AChEs have been calculated via a model that utilizes the structure of T. californica AChE, a spherical ligand, and energy terms that account for electrostatic and van der Waals interactions and chemical potential. These calculations indicate that EA and EI complexes are not bound with respect to electrostatic interactions, which obviates the need for a 'back door' for cationic ligand release. Moreover, the computed energy barriers for ligand release give linear free-energy correlations with log(kE) for substrate turnover, which supports the general correctness of the computational model.
ESTHER : Malany_1999_Chem.Biol.Interact_119-120_99
PubMedSearch : Malany_1999_Chem.Biol.Interact_119-120_99
PubMedID: 10421443

Title : Structure of the Acylation Transition State for Acetylcholinesterase Catalysis -
Author(s) : Malany S , Sikorski RS , Seravalli J , Medhekar R , Quinn DM
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :197 , 1998

Title : Low-Barrier Hydrogen Bond in the Catalytic Triad of Serine Enzymes -
Author(s) : Medhekar R , Baker NA , Sando K , Kearney W , Quinn DM
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :233 , 1998

Title : The Function of Electrostatics in Acetylcholinesterase Catalysis -
Author(s) : Quinn DM , Seravalli J , Nair HK , Medhekar R , Husseini B , Radic Z , Vellom DC , Pickering NA , Taylor P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :203 , 1995