Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems.     

Delta ribozyme has the ability to cleave in transan mRNA

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy.     

Substrate specificity of delta ribozyme cleavage

The specificity of delta ribozyme cleavage was investigated using a trans-acting antigenomic delta ribozyme. Under single turnover conditions, the wild type ribozyme cleaved the 11-mer ribonucleotide substrate with a rate constant of 0.34 min-1, an apparent Km of 17.9 nM and an apparent second-order rate constant of 1.89 x 10(7) min-1 M-1. The substrate specificity of the delta ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of delta ribozyme cleavage.     

Role of the TIGN sequence in E. coli tryptophanyl-tRNA synthetase

Tryptophanyl-tRNA synthetase in E. coli does not have the HIGH sequence that is normally characteristic of class I aminoacyl-tRNA synthetases (EC 6.1.1.2), but instead contains a TIGN sequence at residues 17-20, which has been suggested to be equivalent to the HIGH sequence (Jones, M.D. et al. (1986) Biochemistry 25, 1887-1891). We have overexpressed E. coli Trp-tRNA synthetase and have used site-directed mutagenesis to mutate Thr-17 in the TIGN sequence to alanine. The mutant enzyme has the same Km values as the wild-type for tryptophan or tRNA(Trp), and a slightly increased Km for ATP, from 0.37 to 0.64 mM. On the other hand, the kcat for either the first step or the overall reaction is decreased by a factor of 30. In comparing the Thr-17 and Ala-17 enzymes, the delta delta G for the conversion of substrate to transition state is +9.6 kJ/mol (2.3 kcal/mol). Thr-17 is therefore important in binding the substrate in the transition state, thus supporting the suggestion that TIGN may fulfill the role of a HIGH sequence.     

Thermodynamic dissection of the substrate-ribozyme interaction in the hammerhead ribozyme

The free energy of substrate binding to the hammerhead ribozyme was compared for 10 different hammerheads that differed in the length and sequence of their substrate recognition helices. These hammerheads were selected because neither ribozyme nor substrate oligonucleotide formed detectable alternate secondary structures. The observed free energies of binding varied from -8 to -24 kcal/mol and agreed very well with binding energies calculated from the nearest-neighbor free energies if a constant energetic penalty of DeltaG degreescore = +3.3 +/- 1 kcal/mol is used for the catalytic core. A set of substrates that contained a competing hairpin secondary structure showed weaker binding to the ribozyme by an amount consistent with the predicted free energy for hairpin formation. These thermodynamic conclusions permit the prediction of substrate binding affinities for ribozyme-substrate pairs of any helix length and sequence, and thus, should be very valuable for the rational design of ribozymes directed toward gene inactivation.     

Substrate connectivity effects in the transition state for cytidine deaminase

The binding properties of substrates and competitive inhibitors of Escherichia coli cytidine deaminase are compared with those of the fragments obtained by cutting these ligands at several positions including the glycosidic bond. In contrast with the normal substrate cytidine (kcat/Km = 2.6 x 10(6) M-1 s-1), cytosine is found to serve as an extremely slow substrate (kcat/Km = 1.8 x 10(-3) M-1 s-1), despite the ability of cytosine to enter any active site that can accommodate the normal substrate cytidine. Spontaneous nonenzymatic deamination proceeds at similar rates for cytosine and cytidine at pH 7 and 25 degrees C, indicating that substituent ribose exerts little effect on the intrinsic reactivity of cytidine in solution. Dividing knon by kcat/Km, the maximal Kd value of the enzyme's complex with the altered substrate in the transition state is estimated as 6.1 x 10(-8) M for cytosine, very much higher than the value (1.2 x 10(-16) M) estimated for cytidine. The Kd value of ribofuranose, the missing substituent, is roughly 1.8 x 10(-2) M, as indicated by the Ki values of D-ribose and 1-methyl-D-ribofuranoside as competitive inhibitors. Thus, the free energy of binding of the altered substrate in the transition state is 9.5 kcal/mol more favorable for the whole molecule cytidine than for the sum of those of its parts, cytosine plus ribofuranose. As a separate molecule, however, ribose shows no detectable effect on the enzyme's activity on cytosine. Connectivity effects of similar magnitude are indicated by the equilibrium binding affinities of inhibitors. Thus, the Ki value of the transition state analogue inhibitor zebularine hydrate (1.2 x 10(-12) M) is very much lower than the combined affinities of N-ribofuranosylurea (1.6 x 10(-4) M) and allyl alcohol (0.14 M), indicating that the glycoside bond, by its presence, exerts a connectivity effect of 9.9 kcal/mol on the observed free energy of binding.     

Hammerhead ribozymes targeted to the FBN1 mRNA can discriminate a single base mismatch between ribozyme and target

Hammerhead ribozymes are catalytic RNA molecules that can act in trans, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome. The majority of mutations are single-base changes, many of which exert their effect via a dominant-negative mechanism. Previously we have shown that an antisense hammerhead ribozyme, targeted to the FBN1 mRNA can reduce deposition of fibrillin to the extracellular matrix of cultured fibroblasts, suggesting it may be possible to utilize ribozymes to down regulate the production of mutant protein and thus restore normal fibrillin function. This might be achieved by the mutation creating a ribozyme cleavage site that is not present in the normal allele, however this is likely to limit the number of mutations that could be targeted. Alternatively, it might be possible to target the mutant allele via the ribozyme binding arms. To determine the potential of ribozymes to preferentially target mutant FBN1 alleles via the latter approach, the effect of mismatches in helix I of a hammerhead ribozyme, on the cleavage of fibrillin (FBN1) mRNA was investigated. A single base mismatch significantly reduced ribozyme cleavage efficiency both in vitro and in vivo. The discrimination between fully-matched and mismatched ribozyme varied with the length of helix I, with the discrimination being more pronounced in ribozymes with a shorter helix. These data suggest that it should be possible to design hammerhead ribozymes that can discriminate between closely related (mutant and normal) target RNAs varying in as little as a single nucleotide, even if the mutation does not create a ribozyme cleavage site.     

Effect of an amino acid insertion into the omega loop region of a class C beta-lactamase on its substrate specificity

The extended-substrate specificity of Enterobacter cloacae GC1 beta-lactamase is entirely due to a three amino acid insertion after position 207. To clarify the reason for the extended-substrate specificity, Ala, Ala-Ala, Ala-Ala-Ala, and Ala-Ala-Ala-Ala were inserted after position 207 on the basis of the class C beta-lactamase from E. cloacae P99, respectively. The kcat and Km values of all the mutant enzymes for cephalothin, benzylpenicillin and ampicillin were almost the same as those of the wild-type enzyme, except for those of P99-210-4A which were decreased 4-15-fold. On the other hand, the kcat and Km values for oxyimino beta-lactams such as cefuroxime, ceftazidime, and aztreonam increased with increasing numbers of inserted alanines. The kcat values of the mutant enzymes for cefroxime increased 140-7400-fold compared with that of the wild-type. The Km values also increased with almost the same magnitude, resulting in about the same kcat/Km values as that of the wild-type. On progressive inhibition analysis of aztreonam of the mutant enzymes, two kinds of inactive acyl-enzyme with distinct stabilities were observed, and the proportion of the less stable inactive enzyme increased with increasing numbers of inserted alanines. This suggests that the extension of the substrate specificity is due to instability of the acyl-intermediate caused by an increased deacylation rate in the reaction process.     

Substrate specificities of pepstatin-insensitive carboxyl proteinases from gram-negative bacteria

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 microM, Kcat = 51.4 s-1, and kcat/Km = 8.16 microM-1.s-1. The kinetic parameters for XCP were Km = 3.6 microM, kcat = 52.2 s-1, and kcat/Km = 14.5 microM-1.s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 microM-1.s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 microM-1.s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a Ki value of 2.1 nM, as well as for PCP (Ki = 2.6 nM).     

Ribozymes as therapeutic tools for genetic disease

The discovery that RNA can act as a biological catalyst, as well as a genetic molecule, indicated that there was a time when biological reactions were catalysed in the absence of protein-based enzymes. It also provided the platform to develop those catalytic RNA molecules, called ribozymes, as trans -acting tools for RNA manipulation. Viral diseases or diseases due to genetic lesions could be targeted therapeutically through ribozymes, provided that the sequence of the genetic information involved in the disease is known. The hammerhead ribozyme, one of the smallest ribozymes identified, is able to induce site-specific cleavage of RNA, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Its ability to down-regulate gene expression through RNA cleavage makes the hammerhead ribozyme a candidate for genetic therapy. This could be particularly useful for dominant genetic diseases by down-regulating the expression of mutant alleles. The group I intron ribozyme, on the other hand, is capable of site-specific RNA trans -splicing. It can be engineered to replace part of an RNA with sequence attached to its 3' end. Such application may have importance in the repair of mutant mRNA molecules giving rise to genetic diseases. However, to achieve successful ribozyme-mediated RNA-directed therapy, several parameters including ribozyme stability, activity and efficient delivery must be considered. Ribozymes are promising genetic therapy agents and should, in the future, play an important role in designing strategies for the therapy of genetic diseases.     

The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are "better" substrates. This is compatible with a biological role for the peptidase activity of the enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.     

Ricin A-chain: kinetics, mechanism, and RNA stem-loop inhibitors

Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem-loop structures of 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem-loop RNA which is hydrolyzed at 219 min-1 with a kcat/Km of 4.5 x 10(5) M-1 s-1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower kcat values, but all have Km values of approximately 5 microM. Both the 10- and 18-base substrates have kcat/Km near 10(4) M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem-loop DNA (10 bases) of the same sequence is also a substrate with a kcat/Km of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O-riboside at the depurination site is not a substrate, but binds tightly to the enzyme (Ki = 0.34 microM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pKa of the adenine analogue at N7. Binding of this analogue (Ki = 9.4 microM) is weaker than substrate which indicates that the altered pKa at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (Ki = 0.18 microM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem-loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.     

Mechanisms of cellulases and xylanases: a detailed kinetic study of the exo-beta-1,4-glycanase from Cellulomonas fimi

The exoglucanase/xylanase from Cellulomonas fimi (Cex) has been subjected to a detailed kinetic investigation with a range of aryl beta-D-glycoside substrates. This enzyme hydrolyzes its substrates with net retention of anomeric configuration, and thus it presumably follows a double-displacement mechanism. Values of kcat are found to be invariant with pH whereas kcat/Km is dependent upon two ionizations of pKa = 4.1 and 7.7. The substrate preference of the enzyme increases in the order glucosides < cellobiosides < xylobiosides, and kinetic studies with a range of aryl glucosides and cellobiosides have allowed construction of Broensted relationships for these substrate types. A strong dependence of both kcat (beta 1g = -1) and kcat/Km (beta 1g = -1) upon leaving group ability is observed for the glucosides, indicating that formation of the intermediate is rate-limiting. For the cellobiosides a biphasic, concave downward plot is seenj for kcat, indicating a change in rate-determining step across the series. Pre-steady-state kinetic experiments allowed construction of linear Broensted plots of log k2 and log (k2/Kd) for the cellobiosides of modest (beta 1g = -0.3) slope. These results are consistent with a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states. Secondary deuterium kinetic isotope effects and inactivation experiments provide further insight into transition-state structures and, in concert with beta 1g values, reveal that the presence of the distal sugar moiety in cellobiosides results in a less highly charged transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of methionine-192 of the chymotrypsin active site in the binding and catalysis of mono(amino acid) and peptide substrates

We find that specific oxidation for the Met-192 residue in delta-chymotrypsin to methionine sulfoxide results in a twofold increase in Km(app) and unchanged kcat in the hydrolysis of N-acetyl mono(amino acid) amide substrates. However, the catalyzed hydrolyses of N-acetyl dipeptide amide substrates by (methionine sulfoxide)-192-delta-chymotrypsin (MS-delta-Cht) shows a four- to fivefold decrease in kcat and unchanged Km(app) with respect to delta-chymotrypsin. Hydrolysis of alpha-casein by MS-delta-Cht shows a similar 4.2-fold decrease in kcat. These results imply that the Met-192 acts differently with substrates that bind only in the primary, S1, binding site (i.e., AcPheNH2) from those that bind to more extended regions of the enzyme active site. In the binding of c+AcPheNH2 and AcTrpNH2, the results support a mechanism in which the Met-192 acts to slow the rate of sustrate dissociation from the Michaelis complex to free substrate and enzyme. This is in agreement with the x-ray crystallographic structure of dioxane inhibited alpha-chymotrypsin (Steitz, T., et al. (1969), J. Mol. Biol. 46, 337). However, this mechanism is not apparent when peptide and protein substrates bind. The decrease in kcat on Met-192 modification of approximately fivefold in the hydrolysis of polypeptide substrates show a small, but significant, catalytic contribution of the Met-192 toward the lowering of the energy of activation polypeptide substrate hydrolysis by chymotrypsin. This may support the crystallographic model of Fersht et al. (Fersht, A., et al. (1973), Biochemistry 12, 2035) in which it is proposed that the Met-192 participates in the distortion of bound polypeptide substrates toward the reaction transition-state configuration and, thus, plays a role in catalysis. However, if this mechanism occurs, the effect is small, only contributing about 1 kcal/mol to the lowering of the reaction activation energy.     

Specificity of hammerhead ribozyme cleavage

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.     

Potential roles of conserved amino acids in the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase

The known mammalian 3':5'-cyclic nucleotide phosphodiesterases (PDEs) contain a conserved region located toward the carboxyl terminus, which constitutes a catalytic domain. To identify amino acids that are important for catalysis, we introduced substitutions at 23 conserved residues within the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE; PDE5). Wild-type and mutant proteins were compared with respect to Km for cGMP, kcat, and IC50 for zaprinast. The most dramatic decrease in kcat was seen with H643A and D754A mutants with the decrease in free energy of binding (DeltaDeltaGT) being about 4.5 kcal/mol for each, which is within the range predicted for loss of a hydrogen bond involving a charged residue. His643 and Asp754 are conserved in all known PDEs and are strong candidates to be directly involved in catalysis. Substitutions of His603, His607, His647, Glu672, and Asp714 also produced marked changes in kcat, and these residues are likely to be important for efficient catalysis. The Y602A and E775A mutants exhibited the most dramatic increases in Km for cGMP, with calculated DeltaDeltaGT of 2.9 and 2.8 kcal/mol, respectively, that these two residues are important for cGMP binding in the catalytic site. Zaprinast is a potent competitive inhibitor of cGB-PDE, but the key residues for its binding differ significantly from those that bind cGMP.     

A small, thermostable, and monofunctional chorismate mutase from the archaeon Methanococcus jannaschii

The gene for chorismate mutase (CM) from the archaeon Methanococcus jannaschii, an extreme thermophile, was subcloned and expressed in Escherichia coli. This gene, which belongs to the aroQ class of CMs, encodes a monofunctional enzyme (AroQf) able to complement the CM deficiency of an E. coli mutant strain. The purified protein follows Michaelis-Menten kinetics (kcat = 5.7 s-1 and Km = 41 microM at 30 degreesC) and displays pH-independent activity in the range of pH 5-9. Its activation parameters [Delta H = 16.2 kcal/mol, Delta S = -1. 7 cal/(mol.K)] are similar to those of another well characterized AroQ class CM, the mesophilic AroQp domain from E. coli. Like AroQp, the thermophilic CM is an alpha-helical dimer, but approximately 5 kcal/mol more stable than its mesophilic counterpart as judged from equilibrium denaturation studies. The possible origins of the thermostability of M. jannaschii AroQf, the smallest natural CM characterized to date, are discussed in light of available sequence and tertiary structural information.     

Peptidyl-transferase ribozymes: trans reactions, structural characterization and ribosomal RNA-like features

BACKGROUND: One of the most significant questions in understanding the origin of life concerns the order of appearance of DNA, RNA and protein during early biological evolution. If an 'RNA world' was a precursor to extant life, RNA must be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form amide bonds between RNA and an amino acid or between two amino acids. RESULTS: We characterized peptidyl-transferase reactions catalyzed by two different families of ribozymes that use substrates that mimic A site and P site tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular ('trans') peptide-bond formation, with the two amino-acid substrates binding through an attached AMP or oligonucleotide moiety. CONCLUSIONS: An in vitro-selected ribozyme can catalyze the same type of peptide-bond formation as a ribosome; the ribozyme resembles the ribosome because a very specific RNA structure is required for substrate binding and catalysis, and both amino acids are attached to nucleotides. It is intriguing that, although there are many different possible peptidyl-transferase ribozymes, the sequence and secondary structure of one is strikingly similar to the 'helical wheel' portion of 23S rRNA implicated in ribosomal peptidyl-transferase activity.     

Functional role of the active site glutamate-368 in rat short chain acyl-CoA dehydrogenase

The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids. The degree of overlap in substrate specificity is narrow among these enzymes. The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity. In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD). Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified. The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates. None of the other mutants exhibited enzyme activity. A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified. It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively. These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity.     

Intravenous immunoglobulin in oncology nursing practice

Phenylalanine dehydrogenase from Thermoactinomyces intermedius and leucine dehydrogenase from Bacillus stearothermophilus show a 59% sequence similarity in their substrate-binding domains, although their substrate specificities are different. We prepared a phenylalanine dehydrogenase mutant enzyme whose inherent hexapeptide segment (124F-V-H-A-A-129R) in the substrate-binding domain was replaced by the corresponding part of leucine dehydrogenase (M-D-I-I-Y-Q) in order to investigate the mechanism of substrate recognition by phenylalanine dehydrogenase. The catalytic efficiencies (kcat/Km) of the mutant enzyme with aliphatic amino acids and aliphatic keto acids as substrates were 0.5 to 2% of those of the wild-type enzyme. In contrast, the efficiencies for L-phenylalanine and phenylpyruvate decreased to 0.008 and 0.035% of those of the wild-type enzyme, respectively. These results suggest that the hexapeptide segment plays an important role in the substrate recognition by phenylalanine dehydrogenase.