Active Site Functional Groups Research Articles

A modified group contribution method for estimating thermodynamic parameters of methanol-to-olefins over a SAPO-34 catalyst.

Group contribution (GC) methods, a semi-empirical approach based on the additivity of guest molecular properties, are widely applied to obtain the thermodynamic properties of complex reaction networks. In molecular sieve catalyzed processes, however, the interaction between guest molecules and host active sites also affects thermodynamic properties. In this study, therefore, we propose a modified group contribution (mGC) method by considering the interaction between the groups of guest molecules and independent active site functional groups (IASFGs) in molecular sieves. The mGC method has been used to estimate the thermodynamic properties of guest molecules as well as elementary reactions for the initial stage of methanol to olefins (MTO) reaction over SAPO-34 molecular sieves. It shows that mGC is more accurate than the conventional GC (cGC) methods when compared with the reference data calculated by density functional theory (DFT), indicating that mGC provides an effective way for batch calculation of thermodynamic properties in molecular sieve catalyzed processes.

Identification of 4-acrylamido-N-(pyridazin-3-yl)benzamide as anti-COVID-19 compound: a DFTB, molecular docking, and molecular dynamics study.

Omicron is one of the variants of COVID-19 and continuing member of a pandemic. There are several types of vaccines that were developed around the globe to fight against the virus. However, the world is suffering to find suitable drug candidates for the virus. The main protease (Mpro) enzyme of the virus is the best target for finding drug molecules because of its involvement in viral infection and protein synthesis. ZINC-15 is a database of 750 million commercially available compounds. We find 125 compounds having two aromatic rings and amide groups for non-covalent interactions with active site amino acids and functional groups with the capability to bind –SH group of C145 of Mpro through covalent bonding by a nucleophilic addition reaction. The lead compound (Z144) was identified using molecular docking. The non-covalent interactions (NCI) calculations show the interactions between amino acids present in the active site of the protein and the lead molecules are attractive in nature. The density functional-based tight-binding (DFTB) study of the lead compound with amino acids in the active site indicates that Q190 and Q193 play a very critical role in stabilization. The Michael addition of the acrylamide group of the lead molecule at β-position is facile because the low energy lowest unoccupied molecular orbital (LUMO) is concentrated on the group. From molecular dynamics during 100 ns, it has come to light that strong non-covalent interactions are key for the stability of the lead inside the protein and such binding can fold the protein. The free energy for this interaction is −42.72 kcal mol−1 which was obtained from MM-GB/SA calculations.

Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis.

Acid/base catalysis is an important catalytic strategy used by ribonucleases and ribozymes; however, understanding the number and identity of functional groups involved in proton transfer remains challenging. The proton inventory (PI) technique analyzes the dependence of the enzyme reaction rate on the ratio of D2O to H2O and can provide information about the number of exchangeable sites that produce isotope effects and their magnitude. The Gross-Butler (GB) equation is used to evaluate H/D fractionation factors from PI data typically collected under conditions (i.e., a "plateau" in the pH-rate profile) assuming minimal change in active site residue ionization. However, restricting PI analysis to these conditions is problematic for many ribonucleases, ribozymes, and their variants due to ambiguity in the roles of active site residues, the lack of a plateau within the accessible pL range, or cooperative interactions between active site functional groups undergoing ionization. Here, we extend the integration of species distributions for alternative enzyme states in noncooperative models of acid/base catalysis into the GB equation, first used by Bevilacqua and colleagues for the HDV ribozyme, to develop a general population-weighted GB equation that allows simulation and global fitting of the three-dimensional relationship of the D2O ratio (n) versus pL versus kn/k0. Simulations using the GPW-GB equation of PI results for RNase A, HDVrz, and VSrz illustrate that data obtained at multiple selected pL values across the pL-rate profile can assist in the planning and interpreting of solvent isotope effect experiments to distinguish alternative mechanistic models.

Structural Comparisons of Cefotaximase (CTX-M-ase) Sub Family 1.

The cefotaximase or CTX-M, family of serine-β-lactamases represents a significant clinical concern due to the ability for these enzymes to confer resistance to a broad array of β-lactam antibiotics an inhibitors. This behavior lends CTX-M-ases to be classified as extended spectrum β-lactamases (ESBL). Across the family of CTX-M-ases most closely related to CTX-M-1, the structures of CTX-M-15 with a library of different ligands have been solved and serve as the basis of comparison within this review. Herein we focus on the structural changes apparent in structures of CTX-M-15 in complex with diazabicyclooctane (DABCO) and boronic acid transition state analog inhibitors. Interactions between a positive surface patch near the active site and complementary functional groups of the bound inhibitor play key roles in the dictating the conformations of active site residues. The insights provided by analyzing structures of CTX-M-15 in complex with DABCO and boronic acid transition state analog inhibitors and analyzing existing structures of CTX-M-64 offer opportunities to move closer to making predictions as to how CTX-M-ases may interact with potential drug candidates, setting the stage for the further development of new antibiotics and β-lactamase inhibitors.

Structural insights into inhibitor binding to a fungal ortholog of aspartate semialdehyde dehydrogenase

The aspartate pathway, uniquely found in plants and microorganisms, offers novel potential targets for the development of new antimicrobial drugs. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes production of a key intermediate at the first branch point in this pathway. Several fungal ASADH structures have been determined, but the prior crystallization conditions had precluded complex formation with enzyme inhibitors. The first inhibitor-bound and cofactor-bound structures of ASADH from the pathogenic fungi Blastomyces dermatitidis have now been determined, along with a structural and functional comparison to other ASADH family members. The structure of this new ASADH is similar to the other fungal orthologs, but with some critical differences in the orientation of some active site functional groups and in the subunit interface region. The presence of this bound inhibitor reveals the first details about inhibitor binding interactions, and the flexible orientation of its aromatic ring provides helpful insights into the design of potentially more potent and selective antifungal compounds.

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.

Serendipitous Discovery of α-Hydroxyalkyl Esters as β-Lactamase Substrates

O-(1-Carboxy-1-alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react rapidly with serine β-lactamases, the latter are quite good substrates of representative class A and C, but not D, enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of the α-hydroxy acid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that hydroxamates are also substrates. Structure-activity experiments showed that the α-hydroxyl group was required for any substantial substrate activity. Although both d- and l-α-hydroxy acid derivatives were substrates, the former were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and d-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical substrates. Molecular modeling was employed to explore how the α-hydroxy group might interact with the class C β-lactamase active site. Incorporation of the α-hydroxyalkyl moiety into novel inhibitors will be of considerable interest.

Identification of Selective Enzyme Inhibitors by Fragment Library Screening

The microbial threat to human health is growing due to the dramatic increase in the number of multidrug-resistant organisms. The decline in effective antibiotics available to treat these growing threats has provided greater urgency to the search for new antibiotics. Clearly, new approaches must be developed against novel targets to control these resistant infectious organisms. The screening of low molecular weight compounds against new protein targets provides an opportunity to identify novel inhibitors as starting points for the development of new antibiotics. Custom fragment libraries have been assembled and screened against 3 representative forms of a key enzyme in an essential microbial biosynthetic pathway. Although each of these aspartate semialdehyde dehydrogenases (ASADHs) catalyzes the same reaction and each shares identical active site functional groups, subtle differences in enzyme structures have led to different binding selectivity among the initial hits from these fragment libraries. Amino acid analogues have been identified that show selectivity for either the gram-negative or gram-positive bacterial enzyme forms. A series of benzophenone analogues selectively inhibit the gram-negative ASADH, whereas some haloacids and substituted aromatic acids have been found to inhibit only the fungal form of ASADH. Each of these low molecular weight compounds possesses high ligand binding efficiency for their target enzyme forms. These results support the goal of designing lead compounds that will selectively target ASADHs from different microbial species.

The glmS ribozyme: use of a small molecule coenzyme by a gene-regulatory RNA.

The glmS ribozyme is the first known example of a natural ribozyme that has evolved to require binding of an exogenous small molecule for activity. In Gram-positive bacteria, this RNA domain is part of the messenger RNA (mRNA) encoding the essential enzyme that synthesizes glucosamine-6-phosphate (GlcN6P). When present at physiologic concentration, this small molecule binds to the glmS ribozyme and uncovers a latent self-cleavage activity that ultimately leads to degradation of the mRNA. Biochemical and structural studies reveal that the RNA adopts a rigid fold stabilized by three pseudoknots and the packing of a peripheral domain against the ribozyme core. GlcN6P binding to this pre-organized RNA does not induce conformational changes; rather, the small molecule functions as a coenzyme, providing a catalytically essential amine group to the active site. The ribozyme is not a passive player, however. Active site functional groups are essential for catalysis, even in the presence of GlcN6P. In addition to being a superb experimental system with which to analyze how RNA catalysts can exploit small molecule coenzymes to broaden their chemical versatility, the presence of the glmS ribozyme in numerous pathogenic bacteria make this RNA an attractive target for the development of new antibiotics and antibacterial strategies.

Identification of the Structural Determinants for the Stability of Substrate and Aminoacrylate External Schiff Bases in O-Acetylserine Sulfhydrylase-A

O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the beta-acetoxy group of O-acetyl-L-serine (OAS) by a thiol to give L-cysteine. Previous studies of the K41A mutant enzyme showed L-methionine bound in an external Schiff base (ESB) linkage to PLP as the enzyme was isolated. The mutant enzyme exists in a closed form, optimizing the orientation of the cofactor PLP and properly positioning active site functional groups for reaction. The trigger for closing the active site upon formation of the ESB is thought to be interaction of the substrate alpha-carboxylate with the substrate-binding loop comprised of T68, S69, G70, and N71, and Q142, which is positioned above the cofactor as one looks into the active site. To probe the contribution of these residues to the active site closing and orientation of PLP in the ESB, T68, S69, N71, and Q142 were changed to alanine. Absorbance, fluorescence, near UV-visible CD, and (31)P NMR spectral studies and pre-steady state kinetic studies were used to characterize the mutant enzymes. All of the mutations affect closure of the active site, but to differing extents. In addition, the site appears to be more hydrophilic given that the ESBs do not exhibit a significant amount of the enolimine tautomer. No buildup of the alpha-aminoacrylate intermediate (AA) is observed for the T68A and Q142A mutant enzymes. However, pyruvate is produced at a rate much greater than that of the wild-type enzyme. Data suggest that T68 and Q142 play a role in stabilizing the AA. Both residues donate a hydrogen bond to one of the carboxylate oxygens of the methionine ESB and may also be responsible for the proper orientation of the ESB to generate the AA. The S69A and N71A mutants exhibit formation of the AA, but the rate constant for its formation from the ESB is decreased by 1 order of magnitude compared to that of the wild type. S69 donates a hydrogen bond to the substrate carboxylate in the ESB, while N71 donates hydrogen bonds to O3' of the cofactor and the carboxylate of the ESB; these side chains may also affect the orientation of the ESB. Data suggest that interaction of intermediates with the substrate-binding loop and Q142 gives a properly aligned Michaelis complex and facilitates the beta-elimination reaction.

Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase

Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal structures of as-isolated, functional xanthine oxidase in the course of reaction with the pterin substrate lumazine at 2.2 A resolution and of the nonfunctional desulfo form of the enzyme in complex with xanthine at 2.6 A resolution. In both cases the orientation of substrate is such that the pyrimidine subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed.

Evolution of enzymatic activities in the enolase superfamily: stereochemically distinct mechanisms in two families of cis,cis-muconate lactonizing enzymes.

The mechanistically diverse enolase superfamily is a paradigm for elucidating Nature's strategies for divergent evolution of enzyme function. Each of the different reactions catalyzed by members of the superfamily is initiated by abstraction of the alpha-proton of a carboxylate substrate that is coordinated to an essential Mg(2+). The muconate lactonizing enzyme (MLE) from Pseudomonas putida, a member of a family that catalyzes the syn-cycloisomerization of cis,cis-muconate to (4S)-muconolactone in the beta-ketoadipate pathway, has provided critical insights into the structural bases for evolution of function within the superfamily. A second, divergent family of homologous MLEs that catalyzes anti-cycloisomerization has been identified. Structures of members of both families liganded with the common (4S)-muconolactone product (syn, Pseudomonas fluorescens, gi 70731221 ; anti, Mycobacterium smegmatis, gi 118470554 ) document that the conserved Lys at the end of the second beta-strand in the (beta/alpha)(7)beta-barrel domain serves as the acid catalyst in both reactions. The different stereochemical courses (syn and anti) result from different structural strategies for determining substrate specificity: although the distal carboxylate group of the cis,cis-muconate substrate attacks the same face of the proximal double bond, opposite faces of the resulting enolate anion intermediate are presented to the conserved Lys acid catalyst. The discovery of two families of homologous, but stereochemically distinct, MLEs likely provides an example of "pseudoconvergent" evolution of the same function from different homologous progenitors within the enolase superfamily, in which different spatial arrangements of active site functional groups and substrate specificity determinants support catalysis of the same reaction.

Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

Enzymes are classically proposed to accelerate reactions by binding substrates within active-site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1-1 A scale of most substrate rearrangements. The flexibility of active-site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together 1.2-1.5 A resolution X-ray crystallography, (1)H and (19)F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements to test the distance scale on which noncovalent forces can constrain the structural relaxation or translation of side chains and ligands along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function.

Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis.

Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase.

The activation of the beta-carboxyl group of aspartate catalyzed by aspartokinase is the commitment step to amino-acid biosynthesis in the aspartate pathway. The first structure of a microbial aspartokinase, that from Methanococcus jannaschii, has been determined in the presence of the amino-acid substrate L-aspartic acid and the nucleotide product MgADP. The enzyme assembles into a dimer of dimers, with the interfaces mediated by both the N- and C-terminal domains. The active-site functional groups responsible for substrate binding and specificity have been identified and roles have been proposed for putative catalytic functional groups.

Structure–function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins

RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name "triphosphate tunnel metalloenzyme," is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 5'-end processing.

Intermediates and Transition States in Thiamin Diphosphate-Dependent Decarboxylases. A Kinetic and NMR Study on Wild-Type Indolepyruvate Decarboxylase and Variants Using Indolepyruvate, Benzoylformate, and Pyruvate as Substrates

The thiamin diphosphate (ThDP)-dependent enzyme indolepyruvate decarboxylase (IPDC) is involved in the biosynthetic pathway of the phytohormone 3-indoleacetic acid and catalyzes the nonoxidative decarboxylation of 3-indolepyruvate to 3-indoleacetaldehyde and carbon dioxide. The steady-state distribution of covalent ThDP intermediates of IPDC reacting with 3-indolepyruvate and the alternative substrates benzoylformate and pyruvate has been analyzed by (1)H NMR spectroscopy. For the first time, we are able to isolate and directly assign covalent intermediates of ThDP with aromatic substrates. The intermediate analysis of IPDC variants is used to infer the involvement of active site side chains and functional groups of the cofactor in distinct catalytic steps during turnover of the different substrates. As a result, three residues (glutamate 468, aspartate 29, and histidine 115) positioned perpendicular to the thiazolium moiety of ThDP are involved in binding of all substrates and decarboxylation of the respective tetrahedral ThDP-substrate adducts. Most likely, interactions of these side chains with the substrate-derived carboxylate account for an optimal orientation of the substrate and/or intermediate in the course of carbon-carbon ligation and decarboxylation supporting the suggested least-motion, maximum overlap mechanism. The active site residue glutamine 383, which is located at the opposite site of the thiazolium nucleus as the "carboxylate pocket" (formed by the Glu-Asp-His triad), is central to the substrate specificity of IPDC, probably through orbital alignment. The Glu51-cofactor proton shuttle is, conjointly with the Glu-Asp-His triad, involved in multiple proton transfer steps, including ylide generation, substrate binding, and product release. Studies with para-substituted benzoylformate substrates demonstrate that the electronic properties of the substrate affect the stabilization or destabilization of the carbanion intermediate or carbanion-like transition state and in that way alter the rate dependence on decarboxylation. In conclusion, general mechanistic principles of catalysis of ThDP-dependent enzymes are discussed.

Structural Analysis of Pseudomonas 1-Aminocyclopropane-1-carboxylate Deaminase Complexes: Insight into the Mechanism of a Unique Pyridoxal-5‘-phosphate Dependent Cyclopropane Ring-Opening Reaction,

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable alpha-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an alpha-carbanionic intermediate, which is the common entry for most PLP-dependent reactions, is not obvious. To gain insight into this unusual ring-opening reaction, we have solved the crystal structures of ACC deaminase from Pseudomonas sp. ACP in complex with substrate ACC, an inhibitor, 1-aminocyclopropane-1-phosphonate (ACP), the product alpha-ketobutyrate, and two d-amino acids. Several notable observations of these structural studies include the following: (1) a typically elusive gem-diamine intermediate is trapped in the enzyme complex with ACC or ACP; (2) Tyr294 is in close proximity (3.0 A) to the pro-S methylene carbon of ACC in the gem-diamine complexes, implicating a direct role of this residue in the ring-opening reaction; (3) Tyr294 may also be responsible for the abstraction of the alpha-proton from d-amino acids, a prelude to the subsequent deamination reaction; (4) the steric hindrance precludes accessibility of active site functional groups to the l-amino acid substrates and may account for the stereospecificity of this enzyme toward d-amino acids. These structural data provide evidence favoring a mechanism in which the ring cleavage is induced by a nucleophilic attack at the pro-S beta-methylene carbon of ACC, with Tyr294 as the nucleophile. However, these observations are also consistent with an alternative mechanistic possibility in which the ring opening is acid-catalyzed and may be facilitated by charge relay through PLP, where Tyr294 functions as a general acid. The results of mutagenesis studies corroborated the assigned critical role for Tyr294 in the catalysis.

Evolution of Function in the Crotonase Superfamily: The Stereochemical Course of the Reaction Catalyzed by 2-Ketocyclohexanecarboxyl-CoA Hydrolase

Members of the mechanistically diverse enoyl-CoA hydratase (crotonase) superfamily catalyze reactions that involve stabilization of an enolate anion derived from an acyl thioester of coenzyme A. 2-Ketocyclohexanecarboxyl-CoA hydrolase (BadI), found in a pathway for anaerobic degradation of benzoate by Rhodopseudomonas palustris, is a member of the crotonase superfamily that catalyzes a reverse Dieckmann reaction in which the substrate is hydrolyzed to pimelyl-CoA. The substrate is the configurationally labile 2S-ketocyclohexanecarboxyl-CoA, and in 2H2O solvent hydrogen is incorporated into the 2-proS position of the pimelyl-CoA product. Therefore, the stereochemical course of the BadI-catalyzed reaction is inversion. This information is important for understanding the roles of active-site functional groups in the active site of BadI as well as in the active sites of the homologous 1,4-dihydroxynaphthoyl-CoA synthases that catalyze a forward Dieckmann reaction.

Biochemical detection of adenosine and cytidine ionization within RNA by interference analysis.

Perturbation of active site functional group pKas is an important strategy employed by protein enzymes to achieve catalysis. There is increasing evidence to indicate that RNAs also utilize functional group pKa perturbation for folding and reactivity. The two best candidates for a functionally relevant pKa perturbation are the N3 of C (pKa 4.2) and the N1 of A (pKa 3.5), either of which could be sufficiently raised to allow protonation near physiological pH. Here we report the synthesis and use of a series of alpha-phosphorothioate tagged cytidine and adenosine analogs whose altered pKas make it possible to efficiently detect functionally relevant protonation events by Nucleotide Analog Interference Mapping. This approach has been used to detect ionization events in several catalytic RNAs, including the group I intron, the Hepatitis Delta Virus (HDV), the hairpin and the Varkud Satellite (VS) ribozymes. The active site residue of the VS ribozyme appears to be ionized in the course of the reaction pathway, which may be indicative of a general acid or base mechanism for catalysis by this RNA.