title: Transferase Versus Hydrolase: The Role of Conformational Flexibility in Reaction Specificity.
abstract: Active in the aqueous cellular environment where a massive excess of water is perpetually present, enzymes that catalyze the transfer of an electrophile to a non-water nucleophile (transferases) require specific strategies to inhibit mechanistically related hydrolysis reactions. To identify principles that confer transferase versus hydrolase reaction specificity, we exploited two enzymes that use highly similar catalytic apparatuses to catalyze the transglycosylation (a transferase reaction) or hydrolysis of α-1,3-glucan linkages in the cyclic tetrasaccharide cycloalternan (CA). We show that substrate binding to non-catalytic domains and a conformationally stable active site promote CA transglycosylation, whereas a distinct pattern of active site conformational change is associated with CA hydrolysis. These findings defy the classic view of induced-fit conformational change and illustrate a mechanism by which a stable hydrophobic binding site can favor transferase activity and disfavor hydrolysis. Application of these principles could facilitate the rational reengineering of transferases with desired catalytic properties.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Center for Structural Genomics of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Light
authors.affiliation: Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Laty A
authors.initials: LA
authors.lastname: Cahoon
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Kiran V
authors.initials: KV
authors.lastname: Mahasenan
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Mijoon
authors.initials: M
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Bill
authors.initials: B
authors.lastname: Boggess
authors.affiliation: Department of Biochemistry and Molecular Genetics, Center for Structural Genomics of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
authors.forename: Andrei S
authors.initials: AS
authors.lastname: Halavaty
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Shahriar
authors.initials: S
authors.lastname: Mobashery
authors.affiliation: Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Freitag
authors.affiliation: Department of Biochemistry and Molecular Genetics, Center for Structural Genomics of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Electronic address: wf-anderson@northwestern.edu.
authors.forename: Wayne F
authors.initials: WF
authors.lastname: Anderson
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D020816:Amino Acid Motifs; D001665:Binding Sites; D055162:Biocatalysis; D002236:Carbohydrate Conformation; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D014867:Water; C488014:cycloalternan; D005959:Glucosidases; D006026:Glycoside Hydrolases; C091143:alternanase; C493352:cycloalternan-degrading enzyme, Bacillus
keywords: carbohydrate; hydrolase; non-catalytic binding; protein crystallography; surface binding sites; transferase; transglycosidase; transglycosylase