Andrew J. Bennet

A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites.

BACKGROUND An 18-nucleotide DNA oligomer, PS2.M, derived using an in vitro selection method was previously reported to bind hemin (Fe(III)-protoporphyrinIX) with submicromolar affinity. The DNA-hemin complex exhibited DNA-enhanced peroxidative activity. PS2. M is guanine-rich and requires potassium ions to fold to its active conformation, consistent with its forming a guanine-quaduplex. In investigating the specific catalytic features of PS2.M we tested the peroxidative properties of its RNA version (rPS2.M) as well as that of an unrelated DNA guanine-quadruplex, OXY4. RESULTS The hemin-binding affinity of rPS2.M was found to be 30-fold weaker than that of PS2.M. The UV-visible spectra and kinetics of enzymatic peroxidation of the RNA-hemin complex, however, were nearly identical to those of its DNA counterpart. Both displayed peroxidase activity substantially greater than those of heme proteins such as catalase and Fe(III)-myoglobin. Kinetic analysis suggested that PS2. M and rPS2.M catalyzed the breakdown of the hemin-hydrogen peroxide covalent complex to products. The hemin complex of folded OXY4 (which bound hemin as strongly as did rPS2.M) had a distinct absorption spectrum and only a minor peroxidase activity above the background level. CONCLUSIONS The results indicated that it is possible for RNA and DNA of the same sequence to fold to form comparable cofactor-binding sites, and to show comparable catalytic behavior. The results further suggest that only a subset of cofactor-binding sites formed within folded nucleic acids might be able to function as active sites, by providing the appropriate chemical environments for catalysis.

Guanine-Rich RNAs and DNAs That Bind Heme Robustly Catalyze Oxygen Transfer Reactions

Diverse guanine-rich RNAs and DNAs that fold to form guanine quadruplexes are known to form tight complexes with Fe(III) heme. We show here that a wide variety of such complexes robustly catalyze two-electron oxidations, transferring oxygen from hydrogen peroxide to thioanisole, indole, and styrene substrates. Use of (18)O-labeled hydrogen peroxide reveals the source of the oxygen transferred to form thioanisole sulfoxide and styrene oxide to be the activated ferryl moiety within these systems. Hammett analysis of the kinetics of thioanisole sulfoxide formation is unable to distinguish between a one-step, direct oxygen transfer and a two-step, oxygen rebound mechanism for this catalysis. Oxygen transfer to indole produces a range of products, including indigo and related dyes. Docking of heme onto a high-resolution structure of the G-quadruplex fold of Bcl-2 promoter DNA, which both binds heme and transfers oxygen, suggests a relatively open active site for this class of ribozymes and deoxyribozymes. That heme-dependent catalysis of oxygen transfer is a property of many RNAs and DNAs has ramifications for primordial evolution, enzyme design, cellular oxidative disease, and anticancer therapeutics.

A direct NMR method for the measurement of competitive kinetic isotope effects

We present a technique that uses (13)C NMR spectroscopy to measure kinetic isotope effects on the second-order rate constant (k(cat)/K(m)) for enzyme-catalyzed reactions. Using only milligram quantities of isotopically labeled substrates, precise competitive KIEs can be determined while following the ongoing reaction directly in a NMR spectrometer. Our results for the Vibrio cholerae sialidase-catalyzed hydrolysis of natural substrate analogs support a concerted enzymatic transition state for these reactions.

Galactose recognition by the carbohydrate-binding module of a bacterial sialidase

Glycoside hydrolases often possess carbohydrate-binding modules (CBMs) in addition to their catalytic domains, which help target the enzymes to appropriate substrates and thereby increase their catalytic efficiency. Sialidases hydrolyse the release of sialic acid from a variety of glycoconjugates and play significant roles in the pathogenesis of a number of important diseases. The sialidase from Micromonospora viridifaciens has a CBM which recognizes galactose. The CBM is linked to the catalytic domain by an immunoglobulin-like domain, resulting in the galactose binding site sitting above the catalytic site, suggesting an interplay between the two sites. By studying nine crystallographically independent structures of the M. viridifaciens sialidase, the relative flexibility of the three domains was analysed. A detailed study is also presented of the recognition of galactose and lactose by the M. viridifaciens CBM. The striking structure of this sialidase suggests a role for the CBM in binding to galactose residues unmasked by the adjacent catalytic site.

A stepwise solvent-promoted SNi reaction of α-D-glucopyranosyl fluoride: mechanistic implications for retaining glycosyltransferases.

The solvolysis of α-d-glucopyranosyl fluoride in hexafluoro-2-propanol gives two products, 1,1,1,3,3,3-hexafluoropropan-2-yl α-d-glucopyranoside and 1,6-anhydro-β-D-glucopyranose. The ratio of these two products is essentially unchanged for reactions that are performed between 56 and 100 °C. The activation parameters for the solvolysis reaction are as follows: ΔH(++) = 81.4 ± 1.7 kJ mol(-1), and ΔS(++) = -90.3 ± 4.6 J mol(-1) K(-1). To characterize, by use of multiple kinetic isotope effect (KIE) measurements, the TS for the solvolysis reaction in hexafluoro-2-propanol, we synthesized a series of isotopically labeled α-d-glucopyranosyl fluorides. The measured KIEs for the C1 deuterium, C2 deuterium, C5 deuterium, anomeric carbon, ring oxygen, O6, and solvent deuterium are 1.185 ± 0.006, 1.080 ± 0.010, 0.987 ± 0.007, 1.008 ± 0.007, 0.997 ± 0.006, 1.003 ± 0.007, and 1.68 ± 0.07, respectively. The transition state for the solvolysis reaction was modeled computationally using the experimental KIE values as constraints. Taken together, the reported data are consistent with the retained solvolysis product being formed in an S(N)i (D(N)(++)*A(Nss)) reaction with a late transition state in which cleavage of the glycosidic bond is coupled to the transfer of a proton from a solvating hexafluoro-2-propanol molecule. In comparison, the inverted product, 1,6-anhydro-β-D-glucopyranose, is formed by intramolecular capture of a solvent-equilibrated glucopyranosylium ion, which results from dissociation of the solvent-separated ion pair formed in the rate-limiting ionization reaction (D(N)(++) + A(N)). The implications that this model reaction have for the mode of action of retaining glycosyltransferases are discussed.

Active-site variants of Streptomyces griseus protease B with peptide-ligation activity

BACKGROUND Peptide-ligating technologies facilitate a range of manipulations for the study of protein structure and function that are not possible using conventional genetic or mutagenic methods. To different extents, the currently available enzymatic and nonenzymatic methodologies are synthetically demanding, sequence-dependent and/or sensitive to denaturants. No single coupling method is universally applicable. Accordingly, new strategies for peptide ligation are sought. RESULTS Site-specific variants (Ser195-->Gly, S195G, and Ser195-->Ala, S195A) of Streptomyces griseus protease B (SGPB) were generated that efficiently catalyze peptide ligation (i.e., aminolysis of ester-, thioester- and para-nitroanilide-activated peptides). The variants also showed reduced hydrolytic activity relative to the wild-type enzyme. The ratio of aminolysis to hydrolysis was greater for the S195A variant, which was also capable of catalyzing ligation in concentrations of urea as high as 2 M. CONCLUSIONS Mutagenic substitution of the active-site serine residue of SGPB by either glycine or alanine has created a unique class of peptide-ligating catalysts that are useful for coupling relatively stable ester- and para-nitroanilide-activated substrates. Ligation proceeds through an acyl-enzyme intermediate involving His57. Serine to alanine mutations may provide a general strategy for converting proteases with chymotrypsin-like protein folds into peptide-coupling enzymes.

18 O and secondary 2H kinetic isotope effects confirm the existence of two pathways for acid-catalysed hydrolyses of α-arabinofuranosides

The 18O kinetic isotope effect on the HClO4-catalysed hydrolysis of 4-nitrophenyl [1-18O]-α-arabinofuranoside (k16/k18) is 1.023 ± 0.003 at 80.0 °C; that for isopropyl (1-18O]-α-arabinofuranoside is 0.988 at 30.2 °C and the secondary deuterium effect on the hydrolysis of [2-2H]propan-2-yl α-arabinofuranoside (kH/kD) is 0.979. The nitrophenyl glycoside reacts with exocyclic C–O cleavage and the propan-2-yl glycoside by endocyclic C–O cleavage.

The RNA-Cleaving Bipartite DNAzyme Is a Distinctive Metalloenzyme

Much interest has focused on the mechanisms of the five naturally occurring self‐cleaving ribozymes, which, in spite of catalyzing the same reaction, adopt divergent strategies. These ribozymes, with the exception of the recently described glmS ribozyme, do not absolutely require divalent metal ions for their catalytic chemistries in vitro. A mechanistic investigation of an in vitro‐selected, RNA‐cleaving DNA enzyme, the bipartite, which catalyzes the same chemistry as the five natural self‐cleaving ribozymes, found a mechanism of significant complexity. The DNAzyme showed a bell‐shaped pH profile. A dissection of metal usage indicated the involvement of two catalytically relevant magnesium ions for optimal activity. The DNAzyme was able to utilize manganese(II) as well as magnesium; however, with manganese it appeared to function complexed to either one or two of those cations. Titration with hexaamminecobalt(III) chloride inhibited the activity of the bipartite; this suggests that it is a metalloenzyme that utilizes metal hydroxide as a general base for activation of its nucleophile. Overall, the bipartite DNAzyme appeared to be kinetically distinct not only from the self‐cleaving ribozymes but also from other in vitro‐selected, RNA‐cleaving deoxyribozymes, such as the 8–17, 10–23, and 614.

A potent bicyclic inhibitor of a family 27 α-galactosidase

Two isomeric bicyclo[4.1.0]heptane analogues of the glycosidase inhibitor galacto-validamine, (1R*,2S,3S,4S,5S,6S*)-5-amino-1-(hydroxymethyl)bicyclo[4.1.0]heptane-2,3,4-triol, have been synthesized in 13 steps from 2,3,4,6-tetra-O-benzyl-D-galactose. The inhibitory activities of the two conformationally restricted amines, and their corresponding acetamides, were measured against commercial α-galactosidase enzymes from coffee bean and E. coli. The activity of the glycosyl hydrolase family GH27 enzyme (coffee bean) was competitively inhibited by the 1R,6S-amine (7), a binding interaction that was characterized by a Ki value of 0.541 µM. The GH36 E. coli α-galactosidase exhibited a much weaker binding interaction with the 1R,6S-amine (IC50 = 80 µM). The diastereomeric 1S,6R-amine (9) bound weakly to both galactosidases, (coffee bean, IC50 = 286 µM) and (E. coli, IC50 = 2.46 mM).

Contribution of the Active Site Aspartic Acid to Catalysis in the Bacterial Neuraminidase from Micromonospora Viridifaciens

A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed and purified. Kinetic studies reveal that the replacement of the conserved aspartic acid with glycine results in a catalytically competent retaining sialidase that possesses significant activity against activated substrates. The contribution of this aspartate residue to the free energy of hydrolysis for natural substrates is greater than 19 kJ/mol. The three dimensional structure of the D92G mutant shows that the removal of aspartic acid 92 causes no significant re‐arrangement of the active site, and that an ordered water molecule substitutes for the carboxylate group of D92.