title: Recognition of protein substrates by protein-disulfide isomerase. A sequence of the b' domain responds to substrate binding.
abstract: Refolding of partially folded mitochondrial malate dehydrogenase (mMDH) is assisted by protein-disulfide isomerase (PDI). The addition of a 20-fold molar excess of PDI over denatured protein (0. 1 microM) accelerates the recovery of catalytic activity. PDI fluorescence measurements show that 1 mol of PDI binds 1 mol of denatured mMDH when their concentrations approach 1 microM. The binding of PDI, derivatized with the fluorescence probe iodoacetamide fluorescein, to partially folded mMDH is characterized by a dissociation constant of 0.2 microM. It is shown that the fluorescence probe is covalently attached to a SH residue located in the b' domain. Based on the fluorescence measurements of native and derivatized PDI, it is suggested that recognition of the unfolded substrate involves conformational changes propagated to several domains of PDI.
authors.affiliation: Department of Applied Biology, Hong Kong Polytechnic University, Hong Kong, SAR, China.
authors.forename: P Y
authors.initials: PY
authors.lastname: Cheung
authors.forename: J E
authors.initials: JE
authors.lastname: Churchich
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D018834:Chaperonin 60 / Q000378:metabolism; D004789:Enzyme Activation; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes; D007460:Iodoacetamide / Q000031:analogs & derivatives; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000737:chemistry; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013552:Swine; D014357:Trypsin
chemical_list: D018834:Chaperonin 60; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013439:Sulfhydryl Reagents; D008291:Malate Dehydrogenase; D014357:Trypsin; D000251:Adenosine Triphosphatases; D019704:Protein Disulfide-Isomerases; D007460:Iodoacetamide