title: Kemp Eliminase Activity of Ketosteroid Isomerase.
abstract: Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 109-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.
authors.affiliation: Department of Biochemistry, Stanford University , Stanford, California 94305, United States.
authors.forename: Vandana
authors.initials: V
authors.lastname: Lamba
authors.affiliation: Department of Chemistry and Biochemistry, College of Charleston , Charleston, South Carolina 29424, United States.
authors.forename: Enis
authors.initials: E
authors.lastname: Sanchez
authors.affiliation: Department of Chemistry and Biochemistry, College of Charleston , Charleston, South Carolina 29424, United States.
authors.forename: Lauren Rose
authors.initials: LR
authors.lastname: Fanning
authors.affiliation: Palmetto Homeschool Association , Rock Hill, South Carolina 29730, United States.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Howe
authors.affiliation: R. B. Stall High School , North Charleston, South Carolina 29418, United States.
authors.forename: Maria Alejandra
authors.initials: MA
authors.lastname: Alvarez
authors.affiliation: Department of Biochemistry, Stanford University , Stanford, California 94305, United States.|Department of Chemistry, Department of Chemical Engineering, and Stanford ChEM-H, Stanford University , Stanford, California 94305, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
authors.affiliation: Department of Chemistry and Biochemistry, College of Charleston , Charleston, South Carolina 29424, United States.
authors.forename: Marcello
authors.identifier: 0000-0002-5287-3757
authors.initials: M
authors.lastname: Forconi
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D020604:Comamonas testosteroni / Q000737:chemistry* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D019753:Intramolecular Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007664:Ketosteroids / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D062105:Molecular Docking Simulation; D009154:Mutation; D010080:Oxazoles / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007664:Ketosteroids; D010080:Oxazoles; D011522:Protons; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001120:Arginine; D013251:Steroid Isomerases; D019753:Intramolecular Lyases