title: Exploration of the six tryptophan residues of Escherichia coli cystathionine β-lyase as probes of enzyme conformational change.
abstract: Cystathionine β-lyase (CBL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-homocysteine (l-Hcys), pyruvate and ammonia, in the second step of the transsulfuration pathway of bacteria and plants. A series of 17 site-directed variants of Escherichia coli CBL (eCBL) was constructed to probe the contributions of the six tryptophan residues (W131, W188, W230, W276, W300 and W340) to the fluorescence spectrum of eCBL and to assess their mutability and utility as conformational probes. The effects of these Trp→Phe substitutions on kcat and Km(l)(-Cth) are less than 2-fold, with the exception of the 8-fold increase in Km(l)(-Cth) observed for eCBL-W340F. The midpoint of thermal denaturation, as monitored by circular dichroism spectroscopy, is reduced 4.7°C by the W188F substitution while the targeted replacement of the other five tryptophans alter Tm by less than 1.7°C. The fluorescence spectrum of eCBL is dominated by W230 and the contribution of W340, situated in the active site, is minor. The observed 5-fold increase in the 336 nm fluorescence emission of W188 between 0 and 2M urea, suggests a conformational change at the domain interface. Residues W188 and W340, conserved in proteobacterial CBL enzymes, are situated at the core of the domain interface that forms the active-site cleft. The results of this study suggest that W188 is a useful probe of subtle conformational changes at the domain interface and active site.
authors: Department of Biology, Carleton University, Ottawa K1S 5B6, Canada.
authors: Allison F
authors: AF
authors: Jaworski
authors: Susan M
authors: SM
authors: Aitken
mesh_terms: D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014364:Tryptophan; D008190:Lyases; C019965:cystathionine beta-lyase
keywords: Amino acid metabolism; Cystathionine; Pyridoxal 5′-phosphate; Site-directed mutagenesis; Tryptophan fluorescence