02417nas a2200217   4500008004100000022001400041245016500055210006900220260007300289300001400362490000900376520153500385653003201920653002701952653001901979653001101998653003402009100001602043700001402059856012602073       2006                        eng d  a0304-416500aBiochemical characterization of a low molecular weight aspartic protease inhibitor from thermo-tolerant bacillus licheniformis: kinetic interactions with pepsin0 aBiochemical characterization of a low molecular weight aspartic   aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcDEC  a1845-18560 v17603 a<p>The present article reports a low molecular weight aspartic protease inhibitor, API, from a newly isolated thermo-tolerant Bacillus licheniformis. The inhibitor was purified to homogeneity as shown by rp-HPLC and SDS-PAGE. API is found to be stable over a broad pH range of 2-11 and at temperature 90 degrees C for 2 1/2 h. It has a Mr (relative molecular mass) of 1363 Da as shown by MALDI-TOF spectra and 1358 Da as analyzed by SDS-PAGE The amino acid analysis of the peptide shows the presence of 12 amino acid residues having Mr of 1425Da. The secondary structure of API as analyzed by the CD spectra showed 7% alpha-helix, 49% beta-sheet and 44% aperiodic structure. The Kinetic studies of Pepsin-API interactions reveal that API is a slow-tight binding competitive inhibitor with the IC(50) and K(i) values 4.0nM and (3.83 nM - 5.31 nM) respectively. The overall inhibition constant K(i)* value is 0.107 +/- 0.015 nM. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E+I reversible arrow (k(4), k(5)) EI reversible arrow (k(6), k(7)) EI*. Rate constant k(6) = 2.73 +/- 0.32 s(-1) reveals a fast isomerization of enzyme-inhibitor complex and very slow dissociation as proved by k(7)=0.068 +/- 0.009s(-1). The Rate constants from the intrinsic tryptophanyl fluorescence data is in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI*. (c) 2006 Elsevier B.V. Ail rights reserved.</p>10aAspartic protease inhibitor10aBacillus licheniformis10aenzyme kinetic10aPepsin10aSlow-tight binding inhibition1 aKumar, Ajit1 aRao, Mala  uhttps://library.ncl.res.in/content/biochemical-characterization-low-molecular-weight-aspartic-protease-inhibitor-thermo-102979nas a2200217   4500008004100000022001400041245015900055210006900214260007300283300001200356490000900368520210000377653003202477653002702509653001602536653002002552653003402572100001602606700001402622856012502636       2010                        eng d  a0304-416500aNovel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: kinetic analysis of inhibition and binding affinity0 aNovel bifunctional peptidic aspartic protease inhibitor inhibits  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAY  a526-5360 v18003 a<p>Background: Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniforrnis that inhibits chitinase A (ChiA) from Serratia marcescens. Methods: The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA. Results and conclusions: The peptide has an amino acid sequence N-Ile(1)-Cys(2)-Glu(3)-Ala(4)-Glu(5)-His(6)-Lys(7)-Trp(8)-Gly(9)-Asp(10) -Tyr(11)-Leu(12)-Asp(13)-C. The ChiA-API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I-50= 600 nM and K-i = 510 nM in the presence of chromogenic substrate pnitrophenyl-N,N'-diacetyl-beta-chitobioside[p-NP-(GIcNAc)(2)]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k(5) = 8.7 +/- 1 x 10(-3) s(-1) and k(6) = 7.3 +/- 0.6 x 10(-5) s(-1). CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme. General significance: The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors. (C) 2010 Elsevier B.V. All rights reserved.</p>10aAspartic protease inhibitor10aBacillus licheniformis10aChitinase A10aEnzyme kinetics10aSlow tight binding inhibition1 aKumar, Ajit1 aRao, Mala  uhttps://library.ncl.res.in/content/novel-bifunctional-peptidic-aspartic-protease-inhibitor-inhibits-chitinase-serratia-001448nas a2200193   4500008004100000022001400041245010500055210006900160260007300229300001200302490000700314520068200321653003201003653002501035653003401060100001801094700001401112856012801126       2012                        eng d  a0141-813000aSlow-tight binding inhibition of pepsin by an aspartic protease inhibitor from streptomyces sp MBR040 aSlowtight binding inhibition of pepsin by an aspartic protease i  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcJUL  a165-1740 v513 a<p>The present article reports a low molecular weight aspartic protease inhibitor from a Streptomyces sp. MBR04 exhibiting a two-step inhibition mechanism against pepsin. The kinetic interactions revealed a reversible, competitive, slow-tight binding inhibition with an IC50 and K-i values of 4.5 nM and 4 nM respectively. The conformational changes induced upon inhibitor binding to pepsin was monitored by far and near UV analysis, demonstrated that the inhibitor binds to the active site and causes inactivation. Chemical modification of the inhibitor with WRK and TNBS abolished the antiproteolytic activity of the inhibitor. (c) 2012 Elsevier B.V. All rights reserved.</p>10aAspartic protease inhibitor10aLow molecular weight10aSlow-tight binding inhibition1 aMenon, Vishnu1 aRao, Mala  uhttps://library.ncl.res.in/content/slow-tight-binding-inhibition-pepsin-aspartic-protease-inhibitor-streptomyces-sp-mbr04-0