Theoretical Study of Catalytic Efficiency of a Diels–Alderase Catalytic Antibody: An Indirect Effect Produced During the Maturation Process (original) (raw)
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Reaction mechanisms displayed by catalytic antibodies
Accounts of Chemical Research, 1993
The field of catalytic antibodies now encompasses some 50 examples of various chemical reactions catalyzed by antibodies that have been induced to compounds (haptens) that mimic either the transition states or the high-energy intermediates encountered in those chemical transformations.14 The creation of the "active site" is a consequence of the immunological response to the hapten's structure and will reflect the diversity of solutions to optimizing the molecular interactions between side chain and backbone elements derived from the peptidic antibody framework and complementary regions of the hapten. A central question is the extent to which this response engenders an active site that exhibits the sophisticated catalytic refinements possessed by enzymes that have presumably been optimized by evolution. In this article, we focus on a selected subset of antibody-catalyzed reactions for which there is an appreciable body of evidence for their likely mechanistic course. Although some features clearly are programmed by the inducing hapten, others rich in kinetic and stereochemical complexity appear to arise as a consequence of the chemistry within a confined space walled by protein. Thus, we find and describe reoccurring mechanistic themes displaying characteristics previously associated with enzyme behavior, e.g. multistep substrate processing, induced fit, and allosteric modulation. Instructed by such analogies, we suggest various means for extending the scope and increasing the reactivity of catalytic antibodies (abzymes). Hydrolytic Reactions The ability to cleave peptide bonds site-specifically constitutes an important goal of abzyme research since such catalysts would have many potential applications in controlling biological systems. While amide hydrolysis is a thermodynamically favorable process, the reaction possesses an appreciable kinetic barrier since Jon Stewart, born In Elmlra, NY, In 1984, received both his B.S. and M.S. degrees In chomktry from Buckneli Universlty. He then obtabwd a Ph.D. w e e from Cornell University under the supecvlebn of Professor Bruce Clenem In 1991. He is cwentiy a Helen Hay Whtbwy Foundaflon postdoctoral fellow h Professor Benkovlc's laboratory where he a p p k the tschnlque of protein engineering to catalytic antibodies. He has accepted a faculty posltbn In the D e p a m n t of Chemistry at the University of Florida. Louis J. Lbtta was born In Elizabeth, NJ, In 1963. He earned hls B.S. in chemistry from The Pennsylvania State University In 1985 and his Ph.D. In Organk Chemistry from Corneii University (with B. (ienem) In 1990. He le currently an NIH postdoctoral fellow In the UOratoty of S. J. Benkovlc at The Pennsylvania State University. Ha will join the facuity at Stonehill college, MA. In August 1993. Stephen J. Benkovlc was born In Orange, NJ, and received Me underqaduate degree at Lehlgh and his Ph.D. at Cornell. After a period as a poadoctoral research associate at UCSenta Barbara, he joined the faculty at Penn State Universlty In 1965. He now holds the rank of Evan pueh Profe8.m and the Eberfy Chak In Chemlstry. He has been recognized by numerous natknal and international awards and was elected a member of the National Academy of Sclence in 1985.
A modelling study of a non-concerted hydrolytic cycloaddition reaction by the catalytic antibody H11
Bioorganic & Medicinal Chemistry, 2006
H11 is the first antibody reported to have dual activity as a non-concerted, Diels–Alderase and hydrolytic catalyst. It was previously shown to catalyse the cycloaddition of acetoxybutadiene 1a to N-alkyl maleimides 2 to afford hydroxy-substituted bicyclic adducts 3 with a 30% ee of a major isomer. To better understand this mechanism and the partial stereospecificity, a homology model of H11
Journal of Molecular Biology, 1998
The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was re®ned at a resolution of 1.8 A Ê. The antibody 6D9, which was induced by immunization with the phosphonate transition state analog 3, hydrolyzes a prodrug of chloramphenicol monoester 1 to generate the parent drug 2. The kinetic studies have shown that the antibody is catalytic by virtue of the theoretical relationship between the af®nity for the transition state and the catalytic ef®ciency (k cat /k uncat K S /K TSA). The crystal structure makes it possible to visualize the theoretical relationship. A side-chain (N e) of His L27D is placed in a key position to make a hydrogen bond to the phosphonate oxygen of the transition state analog with a distance of 2.72 A Ê , suggesting a hydrogen bond to the oxyanion developing in the transition state of the hydrolysis. There are no catalytic residues, other than the histidine, around the phosphonate moiety. In addition, in the antibody-hapten complex, the hapten bears a folded conformation and the two stacked aromatic rings are buried deep in the antigen-combining site through aromatic-aromatic interaction with Trp H100I and Tyr H58. The conformation of the bound hapten suggests that the antibody binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable E-form, thereby making it easy for the substrate to reach the transition-state during catalysis. These observations reveal that the catalytic mechanism is explained purely on the basis of the stabilization of the transition state. The re®ned high resolution structures reported here are envisaged to have an impact on the understanding of other hydrolytic antibodies, since their haptens share some unique features with the hapten used in this study.
The Catalytic Mechanism of a Natural Diels–Alderase Revealed in Molecular Detail
Journal of the American Chemical Society, 2016
The Diels-Alder reaction, a [4+2] cycloaddition of a conjugated diene to a dienophile, is one of the most powerful reactions in synthetic chemistry. Biocatalysts capable of unlocking new and efficient Diels-Alder reactions would have major impact. Here we present a molecular-level description of the reaction mechanism of the spirotetronate cyclase AbyU, an enzyme shown here to be a bona fide natural Diels-Alderase. Using enzyme assays, X-ray crystal structures and simulations of the reaction in the enzyme, we reveal how linear substrate chains are contorted within the AbyU active site to facilitate a transannular pericyclic reaction. This study provides compelling evidence for the existence of a natural enzyme evolved to catalyze a Diels-Alder reaction, and shows how catalysis is achieved.
Applied Biochemistry and Biotechnology, 2000
Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Ty p i c a l l y, the choice of TSA s t ru c t u re is s t r a i g h t f o r w a rd, and the criterion for selection in screening is often binding of the TSA to the antibody in a micro t i t e r-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion. The desire d outcome is selective catalysis of formation of the fluorescent derivative in p re f e rence to nonfluorescent side-products. In the study, TSA design was d i rected toward the reaction branch leading to the fluorescent product. Here , we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity at an early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in deriving one or more of the amino acids scre e n e d . In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.
Computational Design of an Enzyme Catalyst for a Stereoselective Bimolecular Diels-Alder Reaction
Science, 2010
The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond forming reactions should be broadly useful in synthetic chemistry.
Molecular Biology, 2017
⎯Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min-1 M-1 for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.