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14: Mechanisms of Catalysis – Serine proteases, Enzyme modification, Ribozymes - Biology

14: Mechanisms of Catalysis – Serine proteases, Enzyme modification, Ribozymes - Biology



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. Chymotrypsin - hydrolyzes peptide bonds on the carboxyl side of aromatic amino acid residues.

B. Mechanism, Chymotrypsin mechanism handout

  1. Catalytic triad forms a charge transfer system to facilitate catalysis
  2. Tetrahedryl intermediate stabilized by enzyme structure, including oxyanion hole
  3. Covalent acyl-enzyme intermediate is relatively stable
  4. Entry of water allows formation of second tetrahedryl intermediate that is similarly stabilized by enzyme
  5. Resolution of second tetrahedryl intermediate restores enzyme and produces second hydrolysis product
  6. Reaction accelerated by approximately a factor of 1010

II. Cofactors - non amino acid compounds that aid catalysis

A. Prosthetic groups - tightly bound to enzyme, e. g. heme and other tetrapyrrols

B. Coenzymes - like prosthetic groups, but not tightly bound so they transiently associate with enzymes, e. coenzyme A, FAD, NAD, NADP

Most Vitamins are precursors to coenzymes. Animals cannot make them and they must be obtained from plants or microbes in animal food.

C. Metal ions: like the iron in heme.

Enzymes have been found that require: Fe, Cu, Zn, Mn, Mg, Co, Ni, Mo, V, Se. This is the reason for organisms requiring trace minerals in their diets. They must have these ions for specific enzymes to function.

III. Ribozymes- catalytic RNA

  1. Ribonuclease P - a ribonucleoprotein ribonuclease where the RNA component is catalytic
  2. Self-splicing ribosomal RNA - an RNA that can both cleave and ligate RNA (acting on itself)
  3. The ribosome is a ribozyme - peptidyl transferase (linking amino acids) occurs in a region of the ribosome where there is only ribosomal RNA
  4. Hypotheses for the early life - the "RNA world"

Contributors

  • Charles S. Gasser (Department of Molecular & Cellular Biology; UC Davis)


Abstract

Background: Research on high-alkaline proteases, such as serine protease PB92, has been largely inspired by their industrial application as protein-degrading components of washing powders. Serine protease PB92 is a member of the subtilase family of enzymes, which has been extensively studied. These studies have included exhaustive protein engineering investigations and X-ray crystallography, in order to provide insight into the mechanism and specificity of enzyme catalysis. Distortions have been observed in the substrate-binding region of subtilisin crystal structures, due to crystal contacts. In addition, the structural variability in the substrate-binding region of subtilisins is often attributed to flexibility. It was hoped that the solution structure of this enzyme would provide further details about the conformation of this key region and give new insights into the functional properties of these enzymes.

Results: The three-dimensional solution structure of the 269-residue (27 kDa) serine protease PB92 has been determined using distance and dihedral angle constraints derived from triple-resonance NMR data. The solution structure is represented by a family of 18 conformers which overlay onto the average structure with backbone and all-heavy-atom root mean square deviations (for the main body of the molecule) of 0.88 and 1.21 Å, respectively. The family of structures contains a number of regions of relatively high conformational heterogeneity, including various segments that are involved in the formation of the substrate-binding site. The presence of flexibility within these segments has been established from NMR relaxation parameters and measurements of amide proton exchange rates.

Conclusions: The solution structure of the serine protease PB92 presents a well defined global fold which is rigid with the exception of a restricted number of sites. Among the limited number of residues involved in significant internal mobility are those of two pockets, termed S1 and S4, within the substrate-binding site. The presence of flexibility within the binding site supports the proposed induced fit mechanism of substrate binding.


Biochemistry. 5th edition.

Protein turnover is an important process in living systems (Chapter 23). Proteins that have served their purpose must be degraded so that their constituent amino acids can be recycled for the synthesis of new proteins. Proteins ingested in the diet must be broken down into small peptides and amino acids for absorption in the gut. Furthermore, as described in detail in Chapter 10, proteolytic reactions are important in regulating the activity of certain enzymes and other proteins.

Proteases cleave proteins by a hydrolysis reaction—the addition of a molecule of water to a peptide bond:

Although the hydrolysis of peptide bonds is thermodynamically favored, such hydrolysis reactions are extremely slow. In the absence of a catalyst, the half-life for the hydrolysis of a typical peptide at neutral pH is estimated to be between 10 and 1000 years. Yet, peptide bonds must be hydrolyzed within milliseconds in some biochemical processes.

The chemical bonding in peptide bonds is responsible for their kinetic stability. Specifically, the resonance structure that accounts for the planarity of a peptide bond (Section 3.2.2) also makes such bonds resistant to hydrolysis. This resonance structure endows the peptide bond with partial double-bond character:

The carbon-nitrogen bond is strengthened by its double-bond character, and the carbonyl carbon atom is less electrophilic and less susceptible to nucleophilic attack than are the carbonyl carbon atoms in compounds such as carboxylate esters. Consequently, to promote peptide-bond cleavage, an enzyme must facilitate nucleophilic attack at a normally unreactive carbonyl group.


Abstract

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pKa of the active site histidine-64 N ε 2 -H was correlated with changes in the second-order rate constant kcat/Km for hydrolysis of tetrapeptide anilides at low ionic strength with a Brønsted slope α = 1.1. The solvent isotope effect D 2 O (kcat/Km)1 = 1.4 ± 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient pxy = ∂βlg/∂pKa (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N−H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure−reactivity parameters.

Atomic coordinates for the S156E/S166D subtilisin variant described in this paper have been deposited in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession code 1Q5P.

Present address: Weill Medical College of Cornell University, 515 East 71st St., Room S-222, New York, NY 10021.

Present address: Roche Biosciences, 3431 Hillview Ave., Palo Alto, CA 94304-1397.


III. Biological Functions of Elastase, Proteinase 3, and Cathepsin G

NSPs are currently viewed as multifunctional enzymes involved in pathogenic agent killing and in inflammatory process regulation (Pham, 2006). First recognized as degradative enzymes able to kill pathogens and cleave extracellular matrix components, NSPs have been attributed a potential role in chemotaxis and migration through cleavage of adhesion molecules at intercellular junctions (Cepinskas et al., 1999 Hermant et al., 2003). This function, however, remains debated because some studies demonstrated decreased neutrophil chemotaxis when using NSP inhibitors (Stockley et al., 1990 Lomas et al., 1995 Young et al., 2007), whereas others have revealed no alteration of neutrophil migration in response to neutrophil-specific chemotactic stimuli in animal models deficient in NSPs (MacIvor et al., 1999 Adkison et al., 2002 Allport et al., 2002).

A. Antimicrobial Roles in Infections

HNE, PR3, and CG participate in direct intracellular killing of phagocytosed bacteria in phagolysosomes in combination with myeloperoxidase and reactive oxygen species generated by the NADPH oxidase complex (Kobayashi et al., 2005). In addition to intracellular killing, extracellular killing can occur through trapping of bacteria in NET composed of filamentous DNA structures decorated with cationic proteases, including NSP secreted by activated neutrophils (Brinkmann et al., 2004). HNE exerts its antimicrobial activity on Gram-negative bacteria by cleaving the outer membrane protein A of Escherichia coli (Belaaouaj et al., 2000) and other virulence factors of Salmonella enterica, Yersinia enterocolitica, and Shigella flexneri (Weinrauch et al., 2002). HNE also prevents the escape of S. flexneri from phagolysosomes in neutrophils (Weinrauch et al., 2002). Extracellular HNE and CG cleave the proinflammatory bacterial virulence factor flagellin (López-Boado et al., 2004) and degrade leukotoxin, a membrane pore-forming virulence factor of the Gram-negative bacteria Actinobacillus actinomycetemcomitans, which can lyse leukocytes and inhibit neutrophil functions (Tsai et al., 1979 Johansson et al., 2000). This pathogen has attracted attention because of its implication in severe destructive periodontal disease. However, the proteolytic activity of NSPs is not necessarily crucial for their antimicrobial activity. Their positive surface charges mediate strong binding to bacterial membranes. This binding alone may inhibit bacterial protein synthesis and induces membrane depolarization and disruption (Zasloff, 2002). Several peptides derived from the CG structure possess antimicrobial properties in vitro (Shafer et al., 1996, 2002). The antimicrobial properties of PR3 are also independent of its protease activity. PR3 efficiently kills both Gram-negative bacteria such as E. coli and Gram-positive bacteria such as Streptococcus faecalis, as well as fungi such as Candida albicans. PR3 also processes human cathelicidin (hCAP18) to its active LL-37 form in the extracellular environment after neutrophil activation (Sørensen et al., 2001). Human cathelicidin is an 18-kDa cationic anti-bacterial protein produced by epithelial cells of the gastrointestinal and respiratory tract and by sebocytes of the skin but also by blood cells, including neutrophils (Doss et al., 2010). Antimicrobial activity requires a proteolytic cleavage of hCAP-18 to liberate the active C-terminal 37-residue polypeptide that exerts broad antimicrobial activity against Gram-negative and -positive bacteria (Dürr et al., 2006).

Mice deficient in NE and CG have been employed as models for exploring their antibacterial functions in microbial infectious diseases. Previous studies showed that mice deficient in NE and CG are more susceptible to infection by Gram-negative and -positive bacteria (Reeves et al., 2002). In neutrophils from NE-deficient [NE(−/−)] mice infected by S. flexneri, 12% of bacteria escaped from the phagolysosome (Weinrauch et al., 2002).

B. Roles in Inflammatory Process Regulation

NSPs are abundantly secreted into the extracellular environment upon neutrophil activation at inflammatory sites. A fraction of the released proteases remain bound in an active form on the external surface of the plasma membrane so that both soluble and membrane-bound NSPs are able to proteolytically regulate the activities of a variety of chemokines, cytokines, growth factors, and cell surface receptors. Secreted proteases also activate lymphocytes and cleave apoptotic and adhesion molecules (Bank and Ansorge, 2001 Pham, 2006 Meyer-Hoffert, 2009). Thus, they retain pro- and anti-inflammatory activities, resulting in a modulation of the immune response at sites of inflammation.

1. Processing of Cytokines, Chemokines, and Growth Factors.

TNF-α and IL-1β are inflammatory cytokines synthesized as inactive membrane-bound pro-forms and are converted to their active forms by the metalloprotease TNF-α converting enzyme (Black et al., 1997) and by the cysteine protease IL-1β-converting enzyme (caspase-1) (Black et al., 1988 Weber et al., 2010), respectively. PR3, however, can also cleave the proforms of TNF-α and IL-1β at Val-Arg bonds (Robache-Gallea et al., 1995 Coeshott et al., 1999). The effect of HNE on the processing of TNF-α is not clear some studies claim that HNE degrades pro-TNF-α with a loss of activity others suggest that HNE liberates soluble, biologically active TNF-α from its membrane-bound precursor. We showed that a synthetic FRET substrate derived from the pro-TNF-α sequence is efficiently cleaved by PR3 and HNE (Korkmaz et al., 2007). PR3, but not HNE, activates IL-1β. This is most probably due to a negatively charged residue at the P4 and a positively charged residue at the P1′ position in pro-IL-1β that electrostatically could interact with Lys99 and Asp 61 of the active-site region of PR3 but not with HNE (Korkmaz et al., 2007).

The pro-inflammatory cytokine IL-18 belongs to the IL-1 cytokine family and is an important regulator of the innate and acquired immune response. It is synthesized as an inactive precursor and can be processed by caspase-1, caspase-3, and PR3 to its active form, but only the caspase-1 and caspase-3 cleavage sites have been identified (Akita et al., 1997). By contrast to HNE, PR3 could cleave caspase 3 substrates because of its preference of aspartyl residues at P2 and P2′ (Korkmaz et al., 2007, 2008c). Caspase 3 cleaves after an Asp residue if another Asp is present at the P4 position (Stennicke et al., 2000 Wei et al., 2000). Thus, PR3 can cleave caspase-3 substrates two residues upstream of the caspase-3 cleavage site if the P2 position of a caspase 3 substrate is compatible with the S1 subsite of PR3. Based on this analysis, the predicted PR3 cleavage site in the pro-IL18 would be at the C-R bond in the -MTD 71 SDC↓RD 76 NA- sequence, whereas that of caspase 3 has been identified after Asp76.

IL-8 is a major chemokine responsible for neutrophil degranulation and neutrophil migration to inflammatory sites (Baggiolini and Clark-Lewis, 1992 Baggiolini et al., 1994). It is secreted as a precursor by neutrophils, monocytes, endothelial cells, and fibroblasts in response to inflammatory stimuli. Full-length pro-IL-8 (77 residues) is converted by MMP-8, MMP-9, and PR3 to three truncated variants (70, 71, and 72 residues long) retaining chemotactic activity in human granulocyte lysates (Padrines et al., 1994 Van den Steen et al., 2000). PR3 (but also lysates of neutrophil granules) cleaves the full-length pro-IL-8 at the Ala-Lys bond (-AVLPRSA 29 ↓KEL-), liberating the most active form of IL-8 (IL-70). The FRET substrate derived from this sequence is cleaved by PR3 but not by HNE (B. Korkmaz, unpublished results). The Ser residue at P2 and positively and negatively charged residues at P1′ and P2′ (bold characters) explain the specific cleavage of pro-IL-8 by PR3.

The stromal cell-derived factor-1α (SDF-1α), also called CXCL12, is a chemokine constitutively produced by hematopoietic cells, but SDF-1 mRNA and protein have also been identified in the central nervous system in neuronal, astroglial, microglial, and endothelial cells. SDF-1α plays a critical role in cell migration because it is a chemotactic factor for T cells, monocytes, pre-B cells, dendritic cells, and hematopoietic progenitor cells (Barbieri et al., 2007). Gene inactivation of SDF-1α or of its receptor CXCR4 in mice impairs myelo- and lymphopoiesis (Ma et al., 1998 Odemis et al., 2005). In vitro, HNE and CG, but not PR3, process the highly flexible N-terminal end of SDF-1α, which results in decreased activity. Processing by HNE releases the N-terminal tripeptide Lys1-Pro2-Val3 (Valenzuela-Fernández et al., 2002), whereas that by CG occurs at the Leu5-Ser6 bond, liberating a pentameric peptide (Delgado et al., 2001).

Progranulin, also called proepithelin, is a heavily glycosylated growth factor of 90 kDa, ubiquitously expressed and involved in tissue regeneration, tumorigenesis, and inflammation (Bateman and Bennett, 1998 Zhu et al., 2002). It is also produced and secreted into the extracellular environment by TNF-α-activated neutrophils. Progranulin inhibits adhesion-dependent neutrophil activation by interfering with the secretion of neutrophil proteases and the production of reactive oxygen species (Zhu et al., 2002), but the mechanisms involved in these anti-inflammatory effects are not yet elucidated. Anti-inflammatory properties of progranulin are suppressed through proteolytic cleavage by HNE and PR3 (Zhu et al., 2002 Kessenbrock et al., 2008). Interestingly enough, processed granulin peptides possess pro-inflammatory properties, stimulating IL-8 release from granulocytes (Zhu et al., 2002). In a recent study, mice lacking both NE and PR3 showed a highly diminished, immune complex-mediated, neutrophil infiltration that was due to impaired progranulin degradation, thus emphasizing the crucial role of progranulin as an inflammation-suppressing mediator (Kessenbrock et al., 2008).

2. Processing and Activation of Cellular Receptors.

NSPs contribute to immune regulation also through the cleavage and activation of specific cellular receptors. HNE, PR3, and CG can process the N-terminal extracellular domains of protease-activated receptors (PARs), which are a subfamily of related G-protein-coupled receptors (Ossovskaya and Bunnett, 2004 Vergnolle, 2009). These receptors are ubiquitously expressed in various tissues and cells and, more especially, in platelets and endothelial cells. Processing of PAR extracellular domains occurs through exposure of a tethered ligand that allows the autoactivation of the receptor and subsequent activation of an intracellular signaling cascade via phospholipase C (Ossovskaya and Bunnett, 2004 Vergnolle, 2009). Four PARs have been identified so far three of them, PAR-1, PAR-3, and PAR-4, can be activated by thrombin. Besides thrombin, CG released from activated neutrophils can also activate PAR-4 at the surface of platelets and initiate their aggregation (Sambrano et al., 2000). All three NSPs cleave PAR-1, which impairs their activation by thrombin (Renesto et al., 1997). The sequence containing the CG cleavage site (-EPF 55 ↓WEDEE-) (Renesto et al., 1997) has been used to raise a sensitive FRET substrate for this protease (Attucci et al., 2002). PAR-2 is expressed on endothelial cells and can be activated by trypsin (SKGR 34 ↓SLIGKV) (Nystedt et al., 1994, 1995a,b) but also by the three NSPs (Uehara et al., 2002, 2003, 2004). PAR-2 activation results in the production and secretion of IL-8 and chemokine (C-C motif) ligand 2 (Uehara et al., 2003, 2004).

Toll-like receptors (TLRs) are transmembrane glycoproteins remarkably contributing to host defense against microbial infections and innate immune response. Thirteen mammalian TLRs have been identified so far. Most of them can recognize specific pathogen-associated molecular patterns presented by invading pathogens or danger-associated molecular patterns released by injured tissues (Akira et al., 2006 Creagh and O'Neill, 2006 Brikos and O'Neill, 2008 Buchanan et al., 2009). The LPS-sensitive TLR4 and EGFR can be directly up-regulated by HNE in lung epithelial cells, which results ultimately in an overproduction of pro-IL-8 mediated through the Myd88/IRAK/TRAF-6 pathway (Walsh et al., 2001 Devaney et al., 2003 Bergin et al., 2008). This HNE effect can be reduced by the addition of the serine protease inhibitor phenylmethylsulfonyl fluoride (Devaney et al., 2003). A new mechanism of HNE-induced pro-IL-8 expression involving activation of the metalloprotease meprin α has been identified (Bergin et al., 2008).

Receptors for the Fc region of IgG and integrin CD11b/CD18 at the neutrophil surface are involved in the recognition of immune complexes, which results in neutrophil activation through modulation of integrin clustering at the cell surface, cytoskeletal rearrangement, and intracellular ROS production (Raptis and Pham, 2005). CG could enhance immune complex-receptor-mediated neutrophil activation as demonstrated using CG/NE double-deficient mice (Raptis et al., 2005), but the relevant CG target has not yet been identified.

3. Induction of Apoptosis by Proteinase 3.

PR3 secreted by activated neutrophils during inflammation is involved in endothelial cell apoptosis (Yang et al., 2001 Preston et al., 2002 Pendergraft et al., 2004). The mechanism by which neutrophil-secreted PR3 enters the endothelial cell has not been elucidated but its molecular intracellular targets have been clearly identified. PR3 directly processes NF㮫 at the Cys-Arg bond in the sequence -KDC 95 ↓RDGA- (Preston et al., 2002) and the cyclin-dependent kinase inhibitor p21 at the Ala-Arg bond in the sequence -IQEA 45 ↓RER- (Dublet et al., 2005) ( Fig. 5 B). The proteolytic processing of NF㮫 and of p21 accelerates endothelial cell apoptosis. Apoptotic elimination of cells from inflammatory sites helps to resolve inflammation. As reported above for pro-IL-18, the positioning of the two Asp residues in the cleaved sequence of NF㮫 indicates that caspase 3 also cleaves this sequence but at a different site, located two residues downstream from the PR3 cleavage site. No preferential HNE cleavage site is present in this sequence, which explains the specific proapoptotic properties of PR3. The mouse homolog of human PR3, however, has a different specificity that impairs the cleavage of NF㮫 and p21 at the same sites (Kalupov et al., 2009) ( Fig. 5 B). Because human and mouse p21 and NF㮫 share the same sequence in this region, it is questionable whether these antiapoptotic properties of PR3 are biologically relevant.

Many other molecular targets of NSPs have been identified as membrane-bound and soluble substrates at inflammatory sites (for review, see Bank and Ansorge, 2001 Wiedow and Meyer-Hoffert, 2005 Pham, 2006, 2008 Meyer-Hoffert, 2009), and many others probably remain to be discovered. Proteolytic cleavage of these molecular targets may result in the activation of latent proforms, in the enhancement or abolishment of pre-existing activities, or in anticipated degradation of inactive proforms that impair interaction with their physiological activators.


Abstract

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pKa of the active site histidine-64 N ε 2 -H was correlated with changes in the second-order rate constant kcat/Km for hydrolysis of tetrapeptide anilides at low ionic strength with a Brønsted slope α = 1.1. The solvent isotope effect D 2 O (kcat/Km)1 = 1.4 ± 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient pxy = ∂βlg/∂pKa (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N−H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure−reactivity parameters.

Atomic coordinates for the S156E/S166D subtilisin variant described in this paper have been deposited in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession code 1Q5P.

Present address: Weill Medical College of Cornell University, 515 East 71st St., Room S-222, New York, NY 10021.

Present address: Roche Biosciences, 3431 Hillview Ave., Palo Alto, CA 94304-1397.


Additional information

Accession codes: Atomic coordinates and structure factors files have been deposited in the Protein Data Bank under accession codes 4RR6, 4RR7, 4RR8, 4RR9, 4RRA, 4RRB, 4RRC, 4RRD, 4RRF, 4RRG, 4RRH, 4RRI, 4RRJ, 4RRK, 4RRL, 4RRM, 4RRQ and 4RRR.

How to cite this article: Ahmed, S. et al. Specificity and catalysis hardwired at the RNA–protein interface in a translational proofreading enzyme. Nat. Commun. 6:7552 doi: 10.1038/ncomms8552 (2015).


Acknowledgements

This work was supported by the Polish Ministry of Science and Higher Education (grant Iuventus Plus IP2012 040172 to M.P.), the Polish National Science Centre (grant 2014/14/M/ST5/00619 to M.D.), and the US National Institutes of Health (grant R01GM099040 to G.S.S.). This project has received founding from the European Union`s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement no. 661187. The Drag laboratory is supported by the Foundation for Polish Science.


Results

3′-S-phosphorothiolate diesters have proven to be useful analogs in the analysis of the catalytic mechanisms used by RNA, protein, and ribonucleoprotein enzymes (Sontheimer 1999). Oxygen and sulfur differ in their abilities to occupy the inner ligand sphere of various metal ions (Sigel et al. 1997 and references therein) for instance, Mg 2+ (a “hard” metal) coordinates well to oxygen, but strongly resists coordination to sulfur. In contrast, “soft” metals such as Mn 2+ , Co 2+ , Zn 2+ , and Cd 2+ readily accept (and in some cases prefer) sulfur as an inner-sphere ligand. For a divalent-metal-dependent reaction that involves a 3′-oxygen as the leaving group (such as splicing), a change in metal specificity from Mg 2+ to a softer metal upon 3′-sulfur substitution implicates a direct metal ion-leaving group interaction.

Group II introns can self-splice by either of two routes—a “branching” or transesterification pathway or a hydrolytic pathway (Fig. A). Both can be relevant in vivo (Podar et al. 1998a) in vitro, either pathway can predominate, depending on the ionic conditions (Daniels et al. 1996). The excised intron is stable in vitro and can catalyze hydrolysis at the exon–exon junction of the spliced product. This spliced exons reopening (SER) reaction is mechanistically analogous to the reversal of the second step of splicing (Podar et al. 1995). To test for the presence of catalytic metal ions in the active site(s) of the group II ribozyme, we used a combination of chemical synthesis (Sun et al. 1997) and enzymatic ligation (Moore and Query 1998) to introduce a 3′-S-phosphorothiolate diester at the site of cleavage for the first (Fig. B) and second (Fig. C) steps of cis-splicing.

3′-Sulfur substitution at the splice sites of a group II intron. (A) The pathways of group II intron self-splicing. Splicing proceeds by either of two pathways, which differ in the identity of the nucleophile during the first step of the reaction. In the branching or transesterification pathway (left), the 2′-hydroxyl group of an adenosine residue attacks the 5′ splice site, giving rise to the exon 1 and lariat intron/exon 2 intermediates. In the hydrolytic pathway (right), water or hydroxide attacks the 5′ splice site, and the intron/exon 2 intermediate is linear. For both pathways, the second step proceeds by attack of the 3′-hydroxyl group of exon 1 on the 3′ splice site, giving rise to spliced exons and releasing the excised intron. As shown at the bottom, released intron can catalyze a SER hydrolytic reaction. (B) 5′ Splice site substitution. A simplified depiction of the yeast mitochondrial group II intron ai5γ is given at the top exons are boxed, and each of the six intron domains is indicated. The bulged A residue that acts as a nucleophile in the first reaction step is shown in domain 6. The sequence of the 5′ splice junction is given underneath [(CS) 3′-thiocytidine]. The structure of the 3′-S-phosphorothiolate linkage at the 5′ splice site is given at the bottom. (C) 3′ Splice site substitution. The diagram is as in B, except that the sequence and the 3′-S-phosphorothiolate linkage are for the 3′ splice site [(US) 3′-thiouridine].

3′-Sulfur substitution at the 5′ splice site of a group II intron results in a metal specificity switch

Self-splicing constructs with a 3′-sulfur substitution (Fig. B) or a normal 3′-oxygen at the 5′ splice site were constructed and tested for cis-splicing activity in vitro (Fig.A) in the presence of 0.5 m (NH4)2SO4 and 100 m m divalent metal ion (Daniels et al. 1996). For the control substrate, no reaction occurred in the absence of divalent metals (Fig. A, lane 4), but exon 1 and spliced product were both generated in the presence of 100 m m MgCl2 (Fig. A, lane 5). When the reactions included 10 or 20 m m MnCl2, CoCl2, ZnCl2, or CdCl2 (Fig. A, lanes 6–13), exon 1 and spliced product were easily detected, indicating that the presence of these metal ions allows efficient splicing (although CoCl2, ZnCl2, and CdCl2 appear to affect the relative rates of the first and second steps, as indicated by the decreased amounts of exon 1 in Fig. A, lanes 8–13). For the 3′-sulfur-containing substrate, no reaction was observed in the absence of divalent metals (Fig. A, lane 17). Unlike the control substrate, however, no reaction was observed in the presence of 100 m m MgCl2 (Fig. A, lane 18). Inclusion of 10 or 20 m m MnCl2, ZnCl2, or CdCl2restored efficient 5′ splice site cleavage (Fig. A, lanes 19, 20, and 23–26), demonstrating a switch in metal specificity for this reaction. CoCl2 (10 or 20 m m ) was unable to restore 5′ splice site cleavage (Fig. A, lanes 21–22). Although MnCl2, ZnCl2, or CdCl2 rescued the first step of the reaction, no spliced product was generated (Fig. A, lanes 19, 20, and 23–26), consistent with the observation that sulfur is a very poor nucleophile at phosphodiester linkages (Pearson 1966Dantzmann and Kiessling 1996).

(A) 3′-Sulfur substitution at the 5′ splice site of a group II intron results in a metal specificity switch. Self-splicing reactions were performed with ligated substrates containing a 3′-oxygen (lanes 1–13) or a 3′-sulfur (lanes 14–26) at the 5′ splice site (see Fig. B). (Lanes 1,14) Unspliced RNAs each substrate was also mock-reacted (lanes 2,15) or reacted (lanes 3,16) with silver(I). Self-splicing reactions were carried out for 1 hr and contained 5 m m EDTA (lanes 4, 17), 100 m m MgCl2 (lanes 5,18), or 10 or 20 m m MnCl2, CoCl2, ZnCl2, or CdCl2 (lanes 6–13, 19–26) as indicated at the top of each lane. Reactions in the presence of MnCl2, CoCl2, ZnCl2, or CdCl2were supplemented with MgCl2 so that the total divalent metal ion concentration was 100 m m . Unspliced precursor (1017 nucleotides), exon 1 intermediate (70 nucleotides), and spliced product (130 nucleotides) are indicated on the left, and the sizes of the DNA markers (in nucleotides) are given on the right. The intron/exon 2 intermediates (lariat and linear) and the corresponding excised intron products contain no radiolabel and are therefore not visible. As an additional standard, lane 27 is a silver cleavage reaction of a 3′-sulfur-containing RNA (79 nucleotides) consisting of exon 1 and only 9 nucleotides of intron sequence. (B) 5′ Splice site cleavage of the 3′-sulfur-substituted substrate occurs accurately in the presence of Mn 2+ . The strategy for mapping the site of cleavage of the modified RNA is given at the top the single 32 P-labeled phosphate group is indicated with an asterisk. Samples were digested with RNase T1, and half of each sample was also treated with iodoacetamide as indicated at the top of each lane. 3′-oxygen and 3′-sulfur products are indicated on the left and right of the gel, respectively. The 8-nucleotide product with a 3′-terminal thiol (lanes 7,9) runs faster than the same fragment with a 3′-terminal hydroxyl (lanes 3,4) because the sulfur is deprotonated under these electrophoresis conditions, adding an extra negative charge.

Treatment of the 3′-sulfur-substituted substrate with silver(I), which induces the specific hydrolysis of the sulfur–phosphorus bond of a 3′-S-phosphorothiolate linkage (Cosstick and Vyle 1990), gave rise to a product of the same size (Fig. A, lane 16), confirming the presence of the 3′-sulfur modification in the substrate and suggesting that 5′ splice site cleavage occurred accurately. Although the 70-nucleotide exon 1 intermediates and the silver-cleaved product comigrated in this 5% polyacrylamide gel, it is possible that the resolution was not sufficient to detect very small size differences (1–2 nucleotides). To confirm the accuracy of 5′ splice site cleavage of the modified substrate, unspliced precursor and the purified products of silver cleavage and Mn 2+ -rescued self-splicing were digested with RNase T1, which cleaves after guanosine residues. Unmodified precursor and exon 1 intermediate were digested in parallel for comparison. We also treated portions of each sample with iodoacetamide (which reacts with thiols but not hydroxyls) to test for the presence of the free 3′-thiol (Weinstein et al. 1996). All samples were then subjected to electrophoresis in a 20% polyacrylamide gel, which provides sufficient resolution to detect single-nucleotide and even single-functional-group differences. As shown in Figure B, the RNase T1-digested products of silver cleavage (lane 7) and self-splicing (lane 9) comigrated precisely. Furthermore, both fragments reacted quantitatively with iodoacetamide, which decreased their mobilities by exactly the same extent (Fig. B, cf. lanes 8 and 10). Iodoacetamide did not react with the fragments derived from unspliced precursors or unmodified exon 1 intermediate (Fig. B, cf. lanes 1, 3, and 5 with lanes 2, 4, and 6, respectively), confirming the specificity of the modification reaction. These results identify the cleavage site as the sulfur-phosphorus bond of the 3′-S-phosphorothiolate linkage. Similar analyses demonstrated the accuracy of 5′ splice site cleavage of the modified substrate in the presence of Zn 2+ and Cd 2+ (data not shown).

To confirm that group II ribozyme activity was required for the observed cleavage in the presence of thiophilic metal ions, we took advantage of a trans reaction characterized by Pyle and coworkers (Fig. , top panel). An RNA consisting of exon 1 and intron domains 1, 2, and 3 (ExD123) has no 5′ splice site cleavage activity on its own, but addition of a separate domain 5 RNA (D5) causes specific 5′ splice site cleavage (Pyle and Green 1994). This reaction appears to be a faithful mimic of the first step of self-splicing (Pyle and Green 1994 Peebles et al. 1995 Podar et al. 1995). We incorporated a 3′-S-phosphorothiolate linkage into the 5′ splice site of an ExD123 RNA and tested the ability of saturating levels of D5 (Pyle and Green 1994) to catalyze the hydrolysis of the sulfur–phosphorus bond. 3′-S-Phosphorothiolate linkages in RNA undergo base-catalyzed breakdown two to three orders of magnitude faster than unmodified phosphodiesters, giving rise to cleavage products with 2′-O,3′-S-cyclic phosphorothiolate and 5′-hydroxyl termini (Liu and Reese 1996 Weinstein et al. 1996). Therefore, the 70-nucleotide exon 1 resulting from either enzymatic hydrolysis or background cleavage differ only in the presence or absence of a 3′-terminal cyclic phosphorothiolate, and cannot be resolved reliably by gel electrophoresis (data not shown). Because the enzymatic reaction is relatively slow (Pyle and Green 1994), the levels of background cleavage are prohibitively high to assay D5-catalyzed hydrolysis directly. Therefore, we treated reaction mixtures with iodoacetamide and RNase T1 to generate fragments that could be resolved from those derived from unreacted or background-cleaved molecules. This assay has the additional advantage of confirming the site of D5-catalyzed 5′ splice site hydrolysis with single-nucleotide accuracy. The reactions are shown in the lower panel of Figure . For the 3′-oxygen control substrate, accurate D5-dependent hydrolysis was observed in 100 m m MgCl2 (Fig. , lane 3), and inclusion of 10 m m MnCl2 (Fig. , lane 5) or 10 m m CdCl2(Fig. , lane 7) did not impair the reaction. [10 m m ZnCl2 is insoluble and causes RNA degradation under the high-KCl conditions of this assay (Pyle and Green 1994) , and therefore could not be tested.] Substitution of the 3′-oxygen leaving group with sulfur blocked the hydrolysis reaction when Mg 2+ was the sole divalent metal ion present (Fig. , cf. lanes 3 and 11). Inclusion of 10 m m MnCl2 (Fig. , lane 13) or CdCl2(Fig. , lane 15) relieved this negative effect. The Mn 2+ - and Cd 2+ -rescued reactions were D5-dependent (Fig. , lanes 12,14) and accurate, as judged by the comigration with a silver-cleaved, iodoacetamide-modified standard (Fig. , lane 9). Therefore, ribozyme activity is required for 5′ splice site cleavage in the presence of these thiophilic divalent metals.

Domain 5 of the group II intron acts intrans to catalyze hydrolysis of the 3′-sulfur-substituted 5′ splice site in a Mn 2+ - or Cd 2+ -dependent manner. The substrate (ExD123) and enzyme (D5) are diagrammed at thetop (the single 32 P-labeled phosphate is denoted as *p). To resolve the product of accurate cleavage from that of background cleavage (see text), all reaction mixtures were treated with iodoacetamide, digested with RNase T1, and subjected to electrophoresis (bottom) as in Fig. B. RNase T1-digested background cleavage product was run off thebottom of the gel. (Lanes 1,8) Unreacted RNAs (lane9) a size standard generated by silver cleavage of the 3′-sulfur-containing substrate. Reactions contained 3 μ m D5 RNA (lanes 3,5,7,11,13,15) and 100 m m MgCl2, 90 m m MgCl2/10 m m MnCl2, or 90 m m MgCl2/10 m m CdCl2 as shown at the top of each lane. Parallel reactions in the absence of D5 RNA (lanes 2,4,6,10,12,14) were done as controls. 3′-Oxygen and 3′-sulfur products are indicated on the left and right of the gel, respectively.

Mg 2+ supports the second reaction step ofcis-splicing of a group II intron containing a 3′-sulfur substitution at the 3′ splice site

To determine whether the second reaction step of self-splicing requires direct coordination of a metal ion to the 3′-oxygen leaving group, we synthesized a self-splicing construct with a 3′-sulfur substitution (Fig. C) or a normal 3′-oxygen at the 3′ splice site, and tested them for cis-splicing activity in vitro in the presence of 0.5 m (NH4)2SO4 and 100 m m divalent metal ion (Fig. A). Separate aliquots of the same reaction were subjected to electrophoresis in 5% (Fig. A, top) and 20% (Fig. A, bottom) polyacrylamide gels. The latter was necessary to visualize the released 10-nucleotide exon 2, generated by the hydrolysis of the exon–exon junction of the spliced product (spliced exons reopening see Fig. A). For the control substrate, no reaction occurred in the absence of divalent metals (Fig. A, lanes 4–7), but intron–exon 2 intermediates (both linear and lariat), spliced product, and released exon 2 were all generated in the presence of 100 m m MgCl2 (Fig. A, lanes 8–11). For the 3′-sulfur-containing substrate, no reaction was observed in the absence of divalent metals (Fig. A, lanes 19–22). In striking contrast to the results obtained with the 5′ splice site, however, 100 m m MgCl2 supported both steps ofcis-splicing (Fig. A, lanes 23–26), as judged by the appearance of lariat intron–exon 2 intermediates, spliced product, and released exon 2. The addition of 10 m m MnCl2 had no significant effect on the reaction rate (data not shown). This asymmetry in the response to 3′-sulfur substitution at the 5′ and 3′ splice sites is exactly what we observed in the spliceosome (Sontheimer et al. 1997). To diminish the unlikely possibility that the second reaction step was supported by contaminating traces of thiophilic metals, we carried out reactions with 110 m m MgCl2 and 10 m m EDTA (Fig. A, lanes 12–15 and 27–30). Because EDTA chelates most thiophilic divalent metal ions five to eight orders of magnitude more tightly than it chelates Mg 2+ (Anderegg 1987), its inclusion would be expected to abolish the ability of trace contaminants to support the reaction. The added EDTA, however, had no effect on the second reaction step with the 3′-sulfur-substituted substrate (Fig. A, cf. lanes 23–26 with lanes 27–30), arguing against this possibility. Treatment of the 3′-sulfur-substituted substrate with silver(I) gave rise to the 10-nucleotide exon 2 fragment (Fig. A, lane 18), confirming the presence of the 3′-sulfur modification in the substrate. Although the high salt present in the self-splicing reactions distorted the electrophoretic mobilities of the exon 2 fragments (Fig. A, lanes 8–15, 23–30), they appeared to comigrate with the silver-cleaved exon 2, providing a preliminary indication that 3′ splice site cleavage occurred accurately.

(A) Mg 2+ supports 3′ splice site cleavage and exon ligation of the 3′-sulfur-substituted substrate. Self-splicing reactions were performed with ligated 3′-end-labeled substrates containing a 3′-oxygen (lanes1–15) or a 3′-sulfur (lanes 16–30) at the 3′ splice site (see Fig. C). Separate aliquots of each sample were subjected to electrophoresis in a 5% polyacrylamide (top) or 20% polyacrylamide (bottom) denaturing gel. (Lanes1,16) Unspliced RNAs each substrate was also mock-reacted (lanes 2,17) or reacted (lanes 3,18) with silver(I). Self-splicing reactions were carried out for 1 hr and contained 10 m m EDTA (lanes 4–7, 19–22), 100 m m MgCl2 (lanes 8–11, 23–26), or 10 m m EDTA mixed with 110 m m MgCl2(lanes 27–30). Samples were incubated for the times (in min) indicated at the top of each lane. Unspliced precursor (967 nucleotides), lariat and linear intron/exon 2 intermediates (897 nucleotides), spliced product (80 nucleotides), and released exon 2 (10 nucleotides) are indicated on the left. The sizes of the DNA markers (in nucleotides) are given on theright. The exon 1 intermediate and the excised intron products (lariat and linear) contain no radiolabel and are therefore not visible. (B) 3′ Splice site cleavage of the 3′-sulfur-substituted substrate occurs accurately in the presence of Mg 2+ . The strategy for mapping the site of cleavage of the modified RNA is given at the top, and is similar to that described in Figure B. The single 32 P-labeled phosphate in the intron is denoted with an asterisk. Samples were digested with RNase T1, and half of each sample was treated with iodoacetamide as indicated at the top of each lane. The products are described on the right the A*pUSH dinucleotide (lanes3,5,7) and its acetamide-modified derivative (lanes4,6,8) are indicated by dots.

To obtain further evidence for the accuracy of 3′ splice site cleavage and exon ligation, we mapped the 3′-terminus of the excised intron RNA directly. The mapping strategy (Fig. B, top) involved incorporation of a single 32 P-labeled phosphate adjacent to the 3′ splice site, RNase T1 digestion, iodoacetamide modification, and comparison with the identically treated product of silver cleavage. If the correct 3′ splice site was used, then RNase T1 digestion should yield the dinucleotide A*pUSH as the only radiolabeled product, and this dinucleotide should be modifiable with iodoacetamide. The data are shown at the bottom of Figure B. Silver cleavage generated the A*pUSH standard (Fig. B, lane 3), which on reaction with iodoacetamide yielded the slower-migrating product A*pUSCH2 C(O)NH2. In the RNase T1 digestions of total RNA from self-splicing reactions in either 100 m m MgCl2 (Fig. B, lane 5) or 90 m m MgCl2/10 m m MnCl2 (Fig. B, lane 7), the A*pUSH dinucleotide is largely obscured by background however, treatment with iodoacetamide clearly generates the identical A*pUSCH2 C(O)NH2 modified dinucleotide in both cases (Fig. B, lanes 6, 8). This product is absent from the RNA derived from unspliced precursor (Fig. B, lane 2). We conclude that the sulfur-phosphorus bond of the 3′ splice site 3′-S-phosphorothiolate linkage is cleaved during self-splicing in the presence of Mg 2+ . Furthermore, the efficiency of accurate 3′ splice site cleavage is not altered by the inclusion of the thiophilic metal Mn 2+ (Fig. B, cf. lanes 6 and 8), providing further evidence against a metal specificity switch during the second step of cis-splicing.

Isolation of the second step of self-splicing uncovers a metal specificity switch

As with the spliceosome (Sontheimer et al. 1997), the ability of Mg 2+ alone to support exon ligation with the 3′-splice-site-substituted substrate could indicate that inner-sphere coordination of the leaving group by a metal ion is not required for the reaction. An alternative possibility, however, is that 3′-sulfur substitution does reduce the rate of the chemical step of exon ligation in the presence of Mg 2+ alone, but this effect is masked by a rate-limiting conformational step (Sontheimer et al. 1997). To distinguish between these possibilities, we assayed the exon ligation reaction in isolation. We took advantage of a recently developed tripartite reaction (A. Bar-Shalom and M. Moore, pers. comm.) in which a 3′ splice site oligonucleotide is added separately to an exon 1 oligonucleotide and a ribozyme containing all but the six 3′-terminal nucleotides of the intron (Fig. A). Unlike other group II exon ligation systems (Podar et al. 1998b Deme et al. 1999), this reaction circumvents the requirement for the inefficient enzymatic ligation step in the construction of the 3′-splice-site-containing substrate. We synthesized and 3′-end-labeled substrate oligonucleotides containing either a 3′-oxygen or a 3′-sulfur at the scissile phosphate and tested them in tripartite exon ligation reactions with an exon 1 oligonucleotide containing a 3′-terminal 2′-deoxycytidine (E1dC) (Fig. B). Although these experiments were done with subsaturating levels of 3′ splice site oligonucleotide (i.e.,k cat/K Mconditions), the rate of the reaction was log-linear with pH (slope ∼1 between pH 5.0 and 6.5), which is consistent with the possibility that the rate is sensitive to the chemical step of the reaction (P.M. Gordon, E.J. Sontheimer, and J.A. Piccirilli, in prep.).

(A) The tripartite step 2 reaction involves attack of an 18-nucleotide exon 1 RNA with a 3′-terminal 2′-deoxycytidine residue (E1dC) on a 13-nucleotide 3′ splice site oligoribonucleotide, catalyzed by an ai5γ ribozyme. The 3′ splice site oligonucleotides contained either a 3′-oxygen or a 3′-sulfur at the scissile phosphate. The products are the 25-nucleotide spliced exons (labeled) and the 6-nucleotide intron fragment (unlabeled). (B) Isolation of the second step of group II self-splicing uncovers a metal specificity switch. Reactions with the 3′-oxygen (lanes 2–23) and 3′-sulfur (lanes25–50) substrates are shown in the top andbottom panels, respectively. Reactions were incubated for the times (in min) given at the top of each lane. Parallel reactions in the absence of ribozyme (lanes16,18,20,22,38–41,43,45,47,49) or the absence of E1dC (lanes15,42) were done as controls. Reactions contained 10 m m EDTA (lanes 3–6, 26–29), 100 m m MgCl2 (lanes 7–10,15–17, 30–33, 43–44), 90 m m MgCl2/10 m m MnCl2 (lanes 11–14, 34–42), 90 m m MgCl2/10 m m CdCl2 (lanes18,19,45,46), 90 m m MgCl2/10 m m CoCl2 (lanes 20,21,47,48), or 90 m m MgCl2/10 m m ZnCl2 (lanes 22,23,49,50).

For the 3′-oxygen control substrate, the reaction in the presence of 100 m m MgCl2 yielded spliced product (Fig. B, lanes 7–10) that comigrated with an independently synthesized and purified standard (Fig. B, lane 1) under conditions where single-nucleotide differences are easily detected. The reaction is dependent on the presence of divalent metal ions (Fig. B, lanes 3–6), E1dC (Fig. B, lane 15), and intron ribozyme (Fig. B, lane 16). Reaction also occurred in the presence of 10 m m MnCl2 (Fig. B, lanes 11–14), although the rate was reduced by approximately twofold. Inclusion of 10 m m CdCl2(Fig. B, lanes 18,19), CoCl2 (Fig. B, lanes 20,21), or ZnCl2 (Fig. B, lanes 22,23) also allowed efficient exon ligation in a ribozyme-dependent manner. For the 3′-sulfur-substituted substrate, no reaction was observed in the absence of divalent metal ions (Fig. B, lanes 26–29), ribozyme (Fig.B, lanes 38–41), or E1dC (Fig. B, lane 42). In the presence of all reaction components, however, the metal ion dependence was very different from that observed in the context ofcis-splicing—reaction in the presence of MgCl2alone (Fig. B, lanes 30–33) resulted in a rate reduction of 100-fold relative to the 3′-oxygen substrate. Inclusion of 10 m m MnCl2 (Fig. B, lanes 34–37) restored the rate to within three- or fourfold of that of the 3′-oxygen substrate under the same conditions (Fig. B, lanes 11–14). Furthermore, 10 m m CdCl2 (Fig. B, lanes 45,46) or CoCl2 (Fig. B, lanes 47,48) also provided strong ribozyme-dependent rate enhancements 10 m m ZnCl2 had only a modest effect (Fig. B, lanes 49,50). Subsequent experiments with saturating amounts of ribozyme showed a similar inhibition in Mg 2+ and rescue in Mn 2+ (data not shown). Therefore, isolation of the second step of self-splicing allowed us to detect a metal ion-leaving group interaction that is obscured during canonical cis-splicing (Fig. A).

Reopening of spliced exons is blocked by 3′-sulfur substitution

Two metal ions have been proposed to catalyze phosphoryl transfer in each step of group II intron self-splicing: one that facilitates deprotonation and activation of the incoming 2′- or 3′-hydroxyl nucleophile, and one that stabilizes the developing negative charge on the oxyanion leaving group (Steitz and Steitz 1993). Although we have provided strong evidence for the latter in both steps of splicing (see above), there is currently no evidence regarding the former. Replacement of an oxygen nucleophile with sulfur is not optimal for the detection of metal-nucleophile interactions because of sulfur’s weak nucleophilicity at phosphate diesters (Dantzmann and Kiessling 1996Pearson 1966). The principle of microscopic reversibility (which states that forward and reverse reactions must proceed through the same transition state) dictates that a metal specificity switch in the reverse reaction is evidence for a metal ion-nucleophile interaction in the forward reaction. Because the SER hydrolytic reaction (Fig. A) is mechanistically analogous to the reverse of the second step of splicing (Podar et al. 1995), 3′-sulfur substitution at the exon–exon junction of the spliced product allows a direct test of the presence of the second metal ion postulated to exist in the group II intron second-step active site (Steitz and Steitz 1993).

We constructed 80-nucleotide spliced exons RNAs with a 3′-sulfur substitution or a normal 3′-oxygen at the exon–exon junction. In the presence of the linear intron ribozyme, MgCl2 supported miscleavage at the two unmodified phosphodiester bonds flanking the exon/exon junction, but no accurately cleaved exon 2 was detected (data not shown). Therefore, substitution of the 3′-oxygen leaving group with sulfur blocks the ability of Mg 2+ to support the accurate SER reaction. We were unable to rescue accurate exon–exon junction hydrolysis, however, despite testing multiple concentrations of many different divalent metal ions (data not shown). Although the loss of activity in Mg 2+ is consistent with the possibility of a direct metal ion interaction, sulfur differs from oxygen in other ways besides metal ion specificity. Accordingly, the absence of rescue by a thiophilic metal means we cannot confidently ascribe the inhibition to the disruption of a metal ion-leaving group interaction, and the proposal for a metal ion-nucleophile interaction in the second step of group II intron self-splicing (Steitz and Steitz 1993) remains tentative.


Mechanisms of catalysis

The favoured model for the enzyme–substrate interaction is the induced fit model. [ 40 ] This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding. These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate that will be altered in the reaction. [ 41 ] Conformational changes can be measured using circular dichroism or dual polarisation interferometry. After binding takes place, one or more mechanisms of catalysis lower the energy of the reaction's transition state by providing an alternative chemical pathway for the reaction. Mechanisms of catalysis include catalysis by bond strain by proximity and orientation by active-site proton donors or acceptors covalent catalysis and quantum tunnelling. [ 30 ] [ 42 ]

Enzyme kinetics cannot prove which modes of catalysis are used by an enzyme. However, some kinetic data can suggest possibilities to be examined by other techniques. For example, a ping–pong mechanism with burst-phase pre-steady-state kinetics would suggest covalent catalysis might be important in this enzyme's mechanism. Alternatively, the observation of a strong pH effect on Vmax but not Km might indicate that a residue in the active site needs to be in a particular ionisation state for catalysis to occur.


Watch the video: Ribozyme structure and activity............. (August 2022).