Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.

The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.

On the mechanism of action of ribonuclease A: relevance of enzymatic studies with a p-nitrophenylphosphate ester and a thiophosphate ester.

It has been reported that His-119 of ribonuclease A plays a major role as an imidazolium ion acid catalyst in the cyclization/cleavage of normal dinucleotides but that it is not needed for the cyclization/cleavage of 3'-uridyl p-nitrophenyl phosphate. We see that this is also true for simple buffer catalysis, where imidazole (as in His-12 of the enzyme), but not imidazolium ion, plays a significant catalytic role with the nitrophenyl substrate, but both are catalytic for normal dinucleotides such as uridyluridine. Rate studies show that the enzyme catalyzes the cyclization of the nitrophenylphosphate derivative 47,000,000 times less effectively (kcat/kuncat) than it does uridyladenosine, indicating that approximately 50% of the catalytic free energy change is lost with this substrate. This suggests that the nitrophenyl substrate is not correctly bound to take full advantage of the catalytic groups of the enzyme and is thus not a good guide to the mechanism used by normal nucleotides. The published data on kinetic effects with ribonuclease A of substituting thiophosphate groups for the phosphate groups of normal substrates has been discussed elsewhere, and it was argued that these effects are suggestive of the classical mechanism for ribonuclease action, not the novel mechanism we have recently proposed. The details of these rate effects, including stereochemical preferences in the thiophosphate series, can be invoked as support for our newer mechanism.

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pKa values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.

An artificial cytochrome P450 that hydroxylates unactivated carbons with regio- and stereoselectivity and useful catalytic turnovers

A catalyst has been synthesized comprising a manganese porphyrin carrying four beta-cyclodextrin groups. It catalyzes the hydroxylation of substrates of appropriate size carrying tert-butylphenyl groups that can hydrophobically bind into the cyclodextrin cavities. In one example as many as 650 catalytic turnovers are seen before the catalyst is oxidatively destroyed, and with a rate comparable to that of typical cytochrome P450 enzymes. In another example, a steroid derivative is regio- and stereoselectively hydroxylated at a single unactivated carbon atom, but more slowly and with fewer turnovers. The carbon attacked is not the most chemically reactive, and the selectivity is determined by the geometry of the catalyst-substrate complex. Nonbinding substrates are not reactive under the conditions used, and substrates with more flexible binding geometries give more than a single product.

Uracil-DNA glycosylase–DNA substrate and product structures: Conformational strain promotes catalytic efficiency by coupled stereoelectronic effects

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil–DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-Å resolution substrate analogue and 2.0-Å resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme–DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

An amino acid as a cofactor for a catalytic polynucleotide

Natural ribozymes require metal ion cofactors that aid both in structural folding and in chemical catalysis. In contrast, many protein enzymes produce dramatic rate enhancements using only the chemical groups that are supplied by their constituent amino acids. This fact is widely viewed as the most important feature that makes protein a superior polymer for the construction of biological catalysts. Herein we report the in vitro selection of a catalytic DNA that uses histidine as an active component for an RNA cleavage reaction. An optimized deoxyribozyme from this selection requires l-histidine or a closely related analog to catalyze RNA phosphoester cleavage, producing a rate enhancement of ≈1-million-fold over the rate of substrate cleavage in the absence of enzyme. Kinetic analysis indicates that a DNA–histidine complex may perform a reaction that is analogous to the first step of the proposed catalytic mechanism of RNase A, in which the imidazole group of histidine serves as a general base catalyst. Similarly, ribozymes of the “RNA world” may have used amino acids and other small organic cofactors to expand their otherwise limited catalytic potential.

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

Beta-Lactamases are widespread in the bacterial world, where they are responsible for resistance to penicillins, cephalosporins, and related compounds, currently the most widely used antibacterial agents. Detailed structural and mechanistic understanding of these enzymes can be expected to guide the design of new antibacterial compounds resistant to their action. A number of high-resolution structures are available for class A beta-lactamases, whose catalytic mechanism involves the acylation of a serine residue at the active site. The identity of the general base which participates in the activation of this serine residue during catalysis has been the subject of controversy, both a lysine residue and a glutamic acid residue having been proposed as candidates for this role. We have used the pH dependence of chemical modification of epsilon-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pH dependence of the epsilon-methylene 1H and 13C chemical shifts (in enzyme selectively labeled with [epsilon-13C]lysine) to estimate the pKa of the relevant lysine residue, lysine-73, of TEM-1 beta-lactamase. Both methods show that the pKa of this residue is > 10, making it very unlikely that this residue could act as a proton acceptor in catalysis. An alternative mechanism in which this role is performed by glutamate-166 through an intervening water molecule is described.

Catalytic editing properties of DNA polymerases.

Enzymatic incorporation of 2',3'-dideoxynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues.