title: Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation.
abstract: A low molecular mass pectate lyase from Fusarium moniliforme was unfolded reversibly by urea and Gdn-HCl at its optimum pH of 8.5, as monitored by intrinsic fluorescence, circular dichroism, and enzymatic activity measurements. Equilibrium unfolding studies yielded a deltaG(H(2)O) of 1.741 kcal/mol, D1/2 of 2.3M, and m value of 0.755kcal/molM with urea and a deltaG(H(2)O) of 1.927kcal/mol, D1/2 of 1.52M, and m value of 1.27 kcal/molM with Gdn-HCl as the denaturant. Thermal denaturation of the pectate lyase at, pH 8.5, was also reversible even after exposure to 75 degrees C for 10 min. Thermodynamic parameters calculated from thermal denaturation curves at pH values from 5.0 to 8.5 yielded a deltaCp of 0.864kcal/(molK). The deltaG(25 degrees C) at, pH 8.5, was 2.06kcal/mol and was in good agreement with the deltaG(H(2)O) values obtained from chemical denaturation curves. There was no exposure of hydrophobic pockets during chemical or thermal denaturation as indicated by the inability of ANS to bind the pectate lyase.
authors.affiliation: Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.
authors.forename: Vaishali S
authors.initials: VS
authors.lastname: Dixit
authors.forename: Ameeta R
authors.initials: AR
authors.lastname: Kumar
authors.forename: Aditi
authors.initials: A
authors.lastname: Pant
authors.forename: M Islam
authors.initials: MI
authors.lastname: Khan
mesh_terms: D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D005670:Fusarium / Q000201:enzymology*; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011133:Polysaccharide-Lyases / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014508:Urea / Q000494:pharmacology; D014867:Water / Q000737:chemistry
chemical_list: D014867:Water; D014508:Urea; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D019791:Guanidine