Catalytic antibodies in healthy humans and patients with autoimmune and viral diseases

Antibodies have been first characterized as proteins produced by the immune system solely for binding other molecules, called antigens, with the goal of eliciting immune response. In this classical conception, antibodies act similarly to enzymes in specific binding to different molecules but cannot catalyze their chemical conversion. However, in 1986 the first monoclonal catalytic antibodies against a chemically stable analog of the transition state of a reaction were obtained and termed abzymes (Abzs). At present, artificial monoclonal Abzs catalyzing more than 100 distinct chemical reactions have been obtained. The discovery of IgG specifically hydrolyzing intestinal vasoactive peptide in the blood serum of asthma patients stimulated studies of natural Abzs. Numerous Abzs discovered afterwards in sera of patients with various autoimmune diseases, viral disorders, or in the milk of healthy mothers, are capable of hydrolyzing proteins, DNA, RNA, polysaccharides, or nucleotides, as well as to phosphorylate proteins and lipids. The phenomenon of catalysis by auto-Abzs is more and more in research focus. In this review we summarize new data on Abzs applications in basic science, medicine and biotechnology.     

Transient oxidation as a mechanistic strategy in enzymatic catalysis

Enzymes that employ a transient oxidation mechanism catalyze transformations that are overall redox neutral, but involve intermediates that have a higher oxidation state than the substrates or products. An oxidation/reduction sequence may be used directly to promote isomerization reactions or indirectly to permit the formation of stabilized intermediates such as carbanions. This review will focus on three recent examples of nicotinamide-dependent enzymes that have been found to employ transient oxidation during catalysis: ADP-L-glycero-D-manno-heptose 6-epimerase, GDP-mannose 3,5-epimerase, and the 6-phosphoglucosidases from family 4. These enzymes are remarkable in their ability to catalyze either nonstereospecific hydride transfers or multiple chemical steps within a single active site.     

Structural basis for antibody catalysis of a disfavored ring closure reaction.

The catalysis of disfavored chemical reactions, especially those with no known natural enzyme counterparts, is one of the most promising achievements of catalytic antibody research. Antibodies 5C8, 14B9, 17F6, and 26D9, elicited by two different transition-state analogues, catalyze disfavored endo-tet cyclization reactions of trans-epoxy alcohols, in formal violation of Baldwin's rules for ring closure. Thus far, neither chemical nor enzyme catalysis has been capable of emulating the extraordinary activity and specificity of these antibodies. X-ray structures of two complexes of Fab 5C8 with the original hapten and with an inhibitor have been determined to 2.0 A resolution. The Fab structure has an active site that contains a putative catalytic diad, consisting of AspH95 and HisL89, capable of general acid/base catalysis. The stabilization of a positive charge that develops along the reaction coordinate appears to be an important factor for rate enhancement and for directing the reaction along the otherwise disfavored pathway. Sequence analysis of the four catalytic antibodies, as well as four inactive antibodies that strongly bind the transition-state analogues, suggests a conserved catalytic mechanism. The occurrence of the putative base HisL89 in all active antibodies, its absence in three out of the four analyzed inactive antibodies, and the rarity of a histidine at this position in immunoglobulins support an important catalytic role for this residue.     

New strategies for the design of catalytic antibodies.

A new approach is advanced which is aimed at expanding the scope of antibody catalysis. It entails the design and synthesis of haptens that will elicit antibodies to catalyze acyl transfer reactions by a methodology which we term "bait and switch" catalysis. What we have done involves the placement of a point charge on the hapten in close proximity to, or in direct substitution for, a chemical functional group we wish to transform in the respective substrate. The haptenic charge should induce a complementary charge (on an amino acid residue) at the binding site. The substrate will lack this charge but will retain a similar overall structure. The monoclonal antibodies that bind these substrates now have the potential to act as general acids/bases for substrates having hydrolyzable functional groups.     

Br?nsted-Evans-Polanyi relationships for C–C bond forming and C–C bond breaking reactions in thiamine-catalyzed decarboxylation of 2-keto acids using density functional theory

The concept of generalized enzyme reactions suggests that a wide variety of substrates can undergo enzymatic transformations, including those whose biotransformation has not yet been realized. The use of quantum chemistry to evaluate kinetic feasibility is an attractive approach to identify enzymes for the proposed transformation. However, the sheer number of novel transformations that can be generated makes this impractical as a screening approach. Therefore, it is essential to develop structure/activity relationships based on quantities that are more efficient to calculate. In this work, we propose a structure/activity relationship based on the free energy of binding or reaction of non-native substrates to evaluate the catalysis relative to that of native substrates. While Br?nsted-Evans-Polanyi (BEP) relationships such as that proposed here have found broad application in heterogeneous catalysis, their extension to enzymatic catalysis is limited. We report here on density functional theory (DFT) studies for C–C bond formation and C–C bond cleavage associated with the decarboxylation of six 2-keto acids by a thiamine-containing enzyme (EC 1.2.7.1) and demonstrate a linear relationship between the free energy of reaction and the activation barrier. We then applied this relationship to predict the activation barriers of 17 chemically similar novel reactions. These calculations reveal that there is a clear correlation between the free energy of formation of the transition state and the free energy of the reaction, suggesting that this method can be further extended to predict the kinetics of novel reactions through our computational framework for discovery of novel biochemical transformations.     

The catalytic power of enzymes: conformational selection or transition state stabilization?

The mechanism by which enzymes produce enormous rate enhancements in the reactions they catalyze remains unknown. Two viewpoints, selection of ground state conformations and stabilization of the transition state, are present in the literature in apparent opposition. To provide more insight into current discussion about enzyme efficiency, a two-state model of enzyme catalysis was developed. The model was designed to include both the pre-chemical (ground state conformations) and the chemical (transition state) components of the process for the substrate both in water and in the enzyme. Although the model is of general applicability, the chorismate to prephenate reaction catalyzed by chorismate mutase was chosen for illustrative purposes. The resulting kinetic equations show that the catalytic power of enzymes, quantified as the k(cat)/k(uncat) ratio, is the product of two terms: one including the equilibrium constants for the substrate conformational states and the other including the rate constants for the uncatalyzed and catalyzed chemical reactions. The model shows that these components are not mutually exclusive and can be simultaneously present in an enzymic system, being their relative contribution a property of the enzyme. The developed mathematical expressions reveal that the conformational and reaction components of the process perform differently for the translation of molecular efficiency (changes in energy levels) into observed enzymic efficiency (changes in k(cat)), being, in general, more productive the component involving the transition state.     

Barrier Compression and Its Contribution to Both Classical and Quantum Mechanical Aspects of Enzyme Catalysis

It is generally accepted that enzymes catalyze reactions by lowering the apparent activation energy by transition state stabilization or through destabilization of ground states. A more controversial proposal is that enzymes can also accelerate reactions through barrier compression—an idea that has emerged from studies of H-tunneling reactions in enzyme systems. The effects of barrier compression on classical (over-the-barrier) reactions, and the partitioning between tunneling and classical reaction paths, have largely been ignored. We performed theoretical and computational studies on the effects of barrier compression on the shape of potential energy surfaces/reaction barriers for model (malonaldehyde and methane/methyl radical anion) and enzymatic (aromatic amine dehydrogenase) proton transfer systems. In all cases, we find that barrier compression is associated with an approximately linear decrease in the activation energy. For partially nonadiabatic proton transfers, we show that barrier compression enhances, to similar extents, the rate of classical and proton tunneling reactions. Our analysis suggests that barrier compression—through fast promoting vibrations, or other means—could be a general mechanism for enhancing the rate of not only tunneling, but also classical, proton transfers in enzyme catalysis.     

Toward bifunctional antibody catalysis

Antibodies that catalyze the deprotonation of unactivated benzisoxazoles to give the corresponding salicylonitriles were prepared using as antigen a 2-aminobenzimidazolium derivative coupled to a carrier protein via its benzene ring. The hapten was designed to induce an antibody binding site with both a base and an acid, in position to initiate proton transfer and stabilize developing negative charge at the phenoxide leaving group, respectively. Consistent with this design, the catalysts exhibit bell-shaped pH-rate profiles, while chemical modification identified several functional groups that could participate in bifunctional catalysis. One of the antibodies, 13G5, is particularly notable in catalyzing the elimination of 6-glutaramidebenzisoxazole with a > 10(5)-fold rate acceleration over background and an effective molarity of > 10(4) M for its catalytic base. These properties compare favorably to the efficiencies achieved by the best previously characterized antibodies with substantially more reactive substrates.     

Managing and manipulating carbocations in biology: terpenoid cyclase structure and mechanism

Terpenoid cyclases catalyze remarkably complex cyclization cascades that are initiated by the formation of a highly reactive carbocation in a polyisoprene substrate. Recent crystal structures of terpenoid cyclases show how these enzymes provide a template for binding and stabilizing the flexible substrate in the precise orientation required for catalysis, trigger carbocation formation, chaperone the conformations of the reactive carbocation intermediates through a unique cyclization sequence, and sequester and stabilize carbocations from premature quenching. Notably, terpenoid cyclases and catalytic antibodies have converged to similar chemical and structural strategies for managing highly reactive carbocations in polyisoprene cyclization cascades.     

Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis

Proteins are intrinsically flexible molecules. The role of internal motions in a protein''s designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.

Author''s Summary

Enzymes are nature''s molecular machines that catalyze biochemical reactions with remarkable efficiency. Recent evidence suggests that enzyme function may involve not only direct structural interactions between the enzyme and its substrate, but also internal motions of the enzyme itself. Here, we describe a computational investigation of three classes of enzymes that catalyze completely different biochemical reactions. Remarkably, the mobile enzyme regions and the nature of these motions are the same across species ranging from single-celled organisms to complex life-forms. Also surprisingly, non-homologous enzymes that catalyze the same chemical reaction but do not share sequence or structural similarity reveal a similar impact of enzyme motions on their reaction mechanisms. Flexible enzyme regions are found to be connected by conserved networks of coupled interactions that connect surface regions to active-site residues. These networks may provide a mechanism for the solvent on an enzyme''s surface to couple to the reaction catalyzed by the enzyme. These results have implications for understanding the mechanism of allostery (long-range effects), and for protein engineering and drug design.     

Transition-state ensemble in enzyme catalysis: possibility, reality, or necessity?

Proteins are not rigid structures; they are dynamic entities, with numerous conformational isomers (substates). The dynamic nature of protein structures amplifies the structural variation of the transition state for chemical reactions performed by proteins. This suggests that utilizing a transition state ensemble to describe chemical reactions involving proteins may be a useful representation. Here we re-examine the nature of the transition state of protein chemical reactions (enzyme catalysis), considering both recent developments in chemical reaction theory (Marcus theory for SN2 reactions), and protein dynamics effects. The classical theory of chemical reactions relies on the assumption that a reaction must pass through an obligatory transition-state structure. The widely accepted view of enzymatic catalysis holds that there is tight binding of the substrate to the transition-state structure, lowering the activation energy. This picture, may, however, be oversimplified. The real meaning of a transition state is a surface, not a single saddle point on the potential energy surface. In a reaction with a "loose" transition-state structure, the entire transition-state region, rather than a single saddle point, contributes to reaction kinetics. Consequently, here we explore the validity of such a model, namely, the enzymatic modulation of the transition-state surface. We examine its utility in explaining enzyme catalysis. We analyse the possibility that instead of optimizing binding to a well-defined transition-state structure, enzymes are optimized by evolution to bind efficiently with a transition-state ensemble, with a broad range of activated conformations. For enzyme catalysis, the key issue is still transition state (ensemble) stabilization. The source of the catalytic power is the modulation of the transition state. However, our definition of the transition state is the entire transition-state surface rather just than a single well-defined structure. This view of the transition-state ensemble is consistent with the nature of the protein molecule, as embodied and depicted in the protein energy landscape of folding, and binding, funnels.     

Enzymatic synthesis of oligosaccharides

Abstract: The biological interest of oligosaccharides is growing very rapidly, and necessitates the development of efficient synthesis reactions. The stereo- and regio-selectivity of enzyme catalysis is a key advantage in this field, as a complementary tool to the chemical approach. Two types of enzymes can be applied to the obtention of oligosaccharides: Hydrolytic enzymes, which can catalyze either reverse hydrolysis (thermodynamic control) or transglycosylation (kinetic control) synthesis reactions; and transferase enzymes, which can use simple carbohydrates from agricultural origin as glycosyl donors.     

Mechanisms of enzyme action and inhibition: Transition state analogues for acid-base catalysis

The theory of absolute reaction rates implies that the grip of a catalyst on a substrate tightens with substrate activation, relaxing later as products are formed and released. Analogs that mimic different kinds of substrate activation can, through the structural details of their complexes with enzymes, indicate how active site residues are involved in the enhancement of reactions rates. In several cases, bonds involved in general acid-base catalysis have been identified tentatively; and recent evidence points to a hydrogen bond of remarkable stability in the transition state in enzymatic deamination of adenosine. Similar approaches have been used to enzymes that act primarily by substrate distortion, nucleophilic catalysis, solvent removal and catalysis by approximation. Two recurring observations, that were not expected in theory, have been the binding of inhibitors in ionized forms that are rare in solution, and changes in enzyme configuration that accompany binding of transition state analogs. Origins and implications of these findings will be discussed with specific reference to the role of solvent water in catalytic phenomena.Supported by Grant GM-18325 from the National Institutes of Health.     

Mechanistic diversity in a metalloenzyme superfamily

It is now appreciated that the relationships of proteins, particularly enzymes, within a protein superfamily can be understood not only in terms of their sequence similarities and three-dimensional structures but also by chemical threads that relate their functional attributes. The mechanistic ties among superfamily members can often be traced to a common transition state for the rate-limiting step of the reactions being catalyzed. This paper presents an analysis of a metalloenzyme superfamily, the members of which catalyze a very diverse set of reactions with unrelated transition states but a more general common mechanistic imperative. The vicinal oxygen chelate (VOC) superfamily is composed of structurally related proteins with paired beta alpha beta beta beta motifs that provide a metal coordination environment with two or three open or readily accessible coordination sites to promote direct electrophilic participation of the metal ion in catalysis. The known types of reactions that are catalyzed include isomerizations (glyoxalase I), epimerizations (methylmalonyl-CoA epimerase), oxidative cleavage of C-C bonds (extradiol dioxygenase), and nucleophilic substitutions (fosfomycin resistance proteins). The remarkable access to mechanism space that is provided by the VOC superfamily appears to derive from a simple, pseudosymmetric structural fold that maximizes the catalytic versatility of the metal center.     

Ligand-protein interaction in biomembrane carriers. The induced transition fit of transport catalysis

Carrier-linked transport through biomembranes is treated under the view of catalysis. As in enzymes, substrate-protein interaction yields catalytic energy in overcoming the activation barrier. At variance with enzymes, catalytic energy is concentrated on structural changes of the carrier rather than on the substrate destabilization for facilitating the global protein rearrangements during transport. A transition state is invoked in which the binding site assumes the best fit to the substrate, whereas in the two ground (internal and external) states, the fit is poor. The maximum binding energy released in the transition state provides catalytic energy to enable the large carrier protein transformations associated with transport. This "induced transition fit" (ITF) of carrier catalysis provides a framework of rules, concerning specificity, unidirectional versus exchange type transport, directing inhibitors to the ground state instead of the transition state, and excluding simultaneous chemical and transport catalysis (vectorial group translocation). The possible role of external energy sources (ATP and Deltapsi) in supplementing the catalytic energy is elucidated. The analysis of the structure-function relationship based on new carrier structures may be challenged to account for the workings of the ITF.     

Biochemistry of terminal deoxynucleotidyltransferase: identification, characterization, requirements, and active-site involvement in the catalysis of associated pyrophosphate exchange and pyrophosphorolytic activity

Terminal deoxynucleotidyltransferase (TdT) has been found to catalyze both pyrophosphate exchange and pyrophosphorolysis reactions. Both reactions are strongly inhibited by antiserum to TdT. The reactions require the presence of a divalent cation, a single- or double-stranded oligomeric or polymeric DNA or RNA, and deoxyribonucleoside triphosphates (for PPi exchange only). Of the three divalent cations tested, Mg2+ and Co2+ are equally effective, while Mn2+ neither is used for catalysis nor inhibits the Mg2+-catalyzed reactions. Ribonucleoside triphosphates have been found to support the PPi exchange reaction to a minor extent and have no inhibitory effect on the catalysis mediated by dNTPs. Inhibition studies, using SH group inhibitors, Zn chelator, and a substrate binding site specific reagent, revealed that PPi exchange and pyrophosphorolysis reactions may be distinguished by differences in their sensitivity to inhibition by various reagents. While the PPi exchange reaction is strongly inhibited by sulfhydryl reagents, o-phenanthroline, and pyridoxal phosphate, the pyrophosphorolysis reaction is insensitive to these reagents. In addition, the pyrophosphorolysis reaction is also found not to require a free 3'-OH terminus of a primer. This difference in the susceptibility of the two reactions indicates that discrete active-site structures exist in TdT which catalyze PPi exchange and pyrophosphorolysis reactions.     

The 2.1 Å crystal structure of an acyl‐CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl‐binding pocket for small branched acyl substrates

The acyl‐AMP forming family of adenylating enzymes catalyze two‐step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X‐ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl‐CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl‐CoA synthetase and 4‐chlorobenzoate:CoA ligase. Most interestingly, the acyl‐binding pocket of the new structure is compared with other acyl‐ and aryl‐CoA synthetases. A comparison of the acyl‐binding pocket of the acyl‐CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl‐binding pocket. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Mechanisms of cytochrome P450 substrate oxidation: MiniReview

Cytochrome P450 (P450) enzymes catalyze a variety of oxidation and some reduction reactions, collectively involving thousands of substrates. A general chemical mechanism can be used to rationalize most of the oxidations and involves a perfenyl intermediate (FeO3+) and odd-electron chemistry, i.e. abstraction of a hydrogen atom or electron followed by oxygen rebound and sometimes rearrangement. This general mechanism can explain carbon hydroxylation, heteroatom oxygenation and dealkylation, epoxidation, desaturation, heme destruction, and other reactions. Another approach to understanding catalysis involves analysis of the more general catalytic cycle, including substrate specificity, because complex patterns of cooperativity are observed with several P450s. Some of the complexity is due to slow conformational changes in the proteins that occur on the same timescale as other steps.     

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.