title: Enzyme Substrate Specificity Conferred by Distinct Conformational Pathways.
abstract: Substrate recognition is one of the hallmarks of enzyme catalysis. Enzyme conformational changes have been linked to selectivity between substrates with little direct evidence. Aldolase, a glycolytic enzyme, must distinguish between two physiologically important substrates, fructose 1-phosphate and fructose 1,6-bisphosphate, and provides an excellent model system for the study of this question. Previous work has shown that isozyme specific residues (ISRs) distant from the active site are responsible for kinetic distinction between these substrates. Notably, most of the ISRs reside in a cluster of five surface α-helices, and the carboxyl-terminal region (CTR), and cooperative interactions among these helices have been demonstrated. To test the hypothesis that conformational changes are at the root of these changes, single surface-cysteine variants were created with the cysteine located on helices of the cluster and CTR. This allowed for site-specific labeling with an environmentally sensitive fluorophore, and subsequent monitoring of conformational changes by fluorescence emission spectrophotometry. These labeled variants revealed different spectra in the presence of saturating amounts of each substrate, which suggested the occurrence of different conformations. Emission spectra collected at various substrate concentrations showed a concentration dependence of the fluorescence spectra, consistent with binding events. Lastly, stopped-flow fluorescence spectrophotometry showed that the rate of these fluorescence changes was on the same time-scale as catalysis, thus suggesting a link between the different fluorescence changes and events during catalysis. On the basis of these results, we propose that different conformational changes may be a common mechanism for dictating substrate specificity in other enzymes with multiple substrates.
authors.affiliation: Program in Biochemistry and Molecular Biology (BMB), Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Florencia
authors.initials: F
authors.lastname: Rago
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine , Boston, Massachusetts 02118, United States .
authors.forename: Daniel
authors.initials: D
authors.lastname: Saltzberg
authors.affiliation: Program in Biochemistry and Molecular Biology (BMB), Boston University , Boston, Massachusetts 02215, United States.|Department of Chemistry, Boston University , Boston, Massachusetts 02215, United States .
authors.forename: Karen N
authors.initials: KN
authors.lastname: Allen
authors.affiliation: Program in Biochemistry and Molecular Biology (BMB), Boston University , Boston, Massachusetts 02215, United States.|Department of Biology, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Dean R
authors.initials: DR
authors.lastname: Tolan
mesh_terms: D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000378:metabolism*; D005635:Fructosediphosphates / Q000737:chemistry / Q000378:metabolism; D005636:Fructosephosphates / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D005635:Fructosediphosphates; D005636:Fructosephosphates; D005634:Fructose-Bisphosphate Aldolase; C029063:fructose-1,6-diphosphate