Transition-metal complexes as alternatives to proteolytic enzymes. Regioselective cleavage of myoglobin by palladium(II) aqua complexes

 The palladium(II) aqua complexes [Pd(H₂O)₄]²⁺, cis-[Pd(en)(H₂O)₂]²⁺, and cis-[Pd(dtco-OH)(H₂O)₂]²⁺ effect hydrolytic cleavage of horse myoglobin in aqueous solution. The conditions were optimized with the third complex. Its structure was determined by X-ray crystallographic analysis of its precursor, the square-planar complex cis-[Pd(dtco-OH)Cl₂], in which the chelating ligand adopts a boat-chair conformation. A weak interaction between the hydroxyl group and the palladium(II) atom seems to improve the stability of the reagent. The yield of cleavage after a 24-h incubation at 60  °C increases from 39% to 85% as the pH decreases from 6.2 to 3.2. The protein fragments are separated by SDS-PAGE electrophoresis and HPLC separation methods, and identified by ESIMS and MALDI-TOF mass spectrometric methods and by determination of terminal amino-acid sequences. Most of the 13 cleavage sites are clustered around the methionine, arginine, and some of the histidine residues, whose side chains can bind to palladium(II). Cleavage tends to occur at the peptide bonds one to three positions removed from the binding residues; the scissile bonds usually lie on the amino-terminal side, seldom on the carboxy-terminal side, of the binding residues. Removal of the heme and unfolding of the protein do not drastically alter the pattern of cleavage. The ability of palladium(II) aqua complexes to cleave proteins at relatively few sites, with explicable selectivity, with good to very good yield, and in weakly acidic and nearly neutral solutions, bodes well for their future use in biochemical and bioanalytical practice. Received: 23 December 1997 / Accepted: 14 April 1998     

Lipoxygenase-mediated cleavage of fatty acids to carbonyl fragments in tomato fruits

Homogenates of tomato fruits catalysed the enzymic conversion of linoleic and linolenic acids (but not oleic acid) to C₆ aldehydes in low (3–5%) molar yield. Hexanal was formed from linoleic acid; cis-3-hexenal and smaller amounts of trans-2-hexenal were formed from linolenic acid. With the fatty acids as substrates, the major products were fatty acid hydroperoxides (50–80% yield) and the ratio of 9- to 13-hydroperoxides as isolated from an incubation with linoleic acid was at least 95:5 in favour of the 9-hydroperoxide isomer. When the 9- and 13-hydroperoxides of linoleic acid were used as substrates with tomato homogenates, the 13-hydroperoxide was readily cleaved to hexanal in high molar yield (60%) but the 9-hydroperoxide isomer was not converted to cleavage products. Properties of the hydroperoxide cleavage system are described. The results indicate that the C₆ aldehydes are formed from C₁₈ polyunsaturated fatty acids in a sequential enzyme system involving lipoxygenase (which preferentially oxygenates at the 9-position) followed by a hydroperoxide cleavage system which is, however, specific for the 13-hydroperoxy isomers.     

Enantioselective cleavage of supercoiled plasmid DNA catalyzed by chiral macrocyclic lanthanide(III) complexes

The enantiomers of the Sm (III), Eu (III) and Yb (III) complexes [LnL(NO₃)₂](NO₃) of a chiral hexaazamacrocycle were tested as catalysts for the hydrolytic cleavage of supercoiled plasmid DNA. The catalytic activity was remarkably enantioselective; while the [LnL^SSSS(NO₃)₂](NO₃) enantiomers promoted the cleavage of plasmid pBR322 from the supercoiled form (SC) to the nicked form (NC), the [LnL^RRRR(NO₃)₂](NO₃) enantiomers were inactive. Kinetics of plasmid DNA hydrolysis was also investigated by agarose electrophoresis and it indicated typical single-exponential cleavage reaction. The hydrolytic mechanism of DNA cleavage was confirmed by the successful ligation of hydrolysis product by T4 ligase. The NMR study of the solutions of the complexes in various buffers indicated that the complexes exist as monomeric cationic complexes [LnL(H₂O)₃]^3 + in slightly acidic solutions and as dimeric cationic complexes [Ln₂L₂(μ-OH)₂(H₂O)₂]^4 + in slightly basic 8 mM solutions, with the latter form being a possible catalyst for hydrolysis of phosphodiester bonds.     

High-yield cleavage of tryptophanyl peptide bonds by o-iodosobenzoic acid.

A new procedure to cleave tryptophanyl peptide bonds in high yield is reported. The method involves treatment of the S-alkylated protein with o-iodosobenzoic acid. The procedure is highly selective for tryptophan and does not modify tyrosine or histidine, but may convert methionine to its sulfoxide derivative. The yields in the cleavage are 70--100%. Tryptophanyl bonds to alanine, glycine, serine, threonine, glutamine, arginine, and S-(pyridylethyl)cysteine are split in nearly quantitative yield, while those preceding isoleucine or valine are split in approximately 70% yield in the proteins examined in this work. The chemical mechanism for tryptophanyl bond cleavage has not been defined, but it is likely that oxidation of the indole ring occurs during the reaction with o-iodosobenzoic acid. Some problems with the quality of commercial preparations of the reagent are discussed.     

Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide.

The four major internal structural proteins (the group-specific antigens) of avian myeloblastosis virus are formed by sequential cleavage of a precursor polypeptide with M_r = 76,000 (Pr76). The evidence for this conclusion is based on analysis of immune precipitates from lysates of AMV⁴-infected cells treated with a multivalent antiserum directed against these proteins. Sodium dodecyl sulfate gel electrophoresis of such immune precipitates from cells pulse-labeled with [³⁵S]-methionine reveals five metabolically unstable radioactive polypeptides. These polypeptides behave kinetically as precursors to virion proteins. Double-label ion-exchange chromatography of tryptic digests of the unstable polypeptides demonstrates that the largest precursor, Pr76, contains the amino acid sequences of all four virion proteins. It appears not to contain the sequence of the fifth and smallest internal virion protein. The four smaller precursors are intermediate cleavage products of Pr76.The arrangement of the virion proteins in Pr76 was determined by labeling cells shortly after inhibiting polypeptide chain initiation. The relative amounts of radioactivity both in completed virion proteins and in the tryptic peptides of Pr76 implies the same order for three of the four proteins. The exact position of one protein remains uncertain.On the basis of these experiments, we propose a cleavage pathway for the generation of the structural proteins of AMV. We also demonstrate that cleavage of precursors can proceed in crude extracts of AMV-infected cells. This proteolysis, while resistant to several protease inhibitors, is completely blocked by addition of agents that disrupt membranes.     

Suberoylanilide hydroxamic acid induces ROS-mediated cleavage of HSP90 in leukemia cells

Heat shock protein 90 (HSP90) is a molecular chaperone that supports stability of client proteins. We found that HSP90 was cleaved to 55 kDa protein after treatment with histone deacetylase (HDAC) inhibitors including suberoylanilide hydroxamic acid (SAHA) in several leukemia cell lines. We further analyzed molecular changes induced by SAHA in K562 cells. The SAHA-induced cleavage of HSP90 was blocked by a pan-caspase inhibitor, z-VAD-fmk, implying that the process is dependent on caspase activity. However, the experiments using antagonistic and agonistic Fas antibodies revealed that the cleavage of HSP90 was not dependent on Fas signaling. SAHA induced generation of reactive oxygen species (ROS), and the cleavage of HSP90 was blocked by a ROS scavenger N-acetylcystein (NAC). We also confirmed that hydrogen peroxide (H₂O₂) induced cleavage of HSP90 in a similar manner. Caspase 2, 3, 4, 6, 8, and 10 were activated by treatment with SAHA, and the activities were reduced by the pretreatment of NAC. Treatment of the cells with caspase 10 inhihitor, but not other inhibitors of caspases activated by SAHA, prevented cleavage of HSP90 by SAHA. SAHA-induced ROS generation and HSP90 cleavage were dependent on newly synthesized unknown proteins. Taken together, our results suggest that the cleavage of HSP90 by SAHA is mediated by ROS generation and caspase 10 activation. HSP90 cleavage may provide an additional mechanism involved in anti-cancer effects of HDAC inhibitors.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0533-4) contains supplementary material, which is available to authorized users.     

Degradation of purified neurofilament subunits by calcium-activated neutral protease: characterization of the cleavage products

Neurofilament proteins (NFP) purified from rat spinal cord were labeled with ¹²⁵-I and incubated with a crude extract from rat spinal cord containing Ca²⁺-activated protease(s). The protease(s) activated by mM Ca²⁺ cleaved the NFP and produced a series of breakdown products which were different for each NFP. The amount of cleavage was dependent upon the incubation time with proteases but the pattern remained constant. Some of the cleavage products were relatively stable. These observations suggest that the cleavage products produced by treating NFP subunits with the endogenous protease can be used as a finger print to further study NFP metabolism and to better understand their role in physiological and pathological conditions of the nervous system.     

The reversibility of adenosine triphosphate cleavage by myosin

For the simplest kinetic model the reverse rate constants (k₋₁ and k₋₂) associated with ATP binding and cleavage on purified heavy meromyosin and heavy meromyosin subfragment 1 from rabbit skeletal muscle in the presence of 5mm-MgCl₂, 50mm-KCl and 20mm-Tris–HCl buffer at pH8.0 and 22°C are: k₋₁<0.02s⁻¹ and k₋₁=16s⁻¹. Apparently, higher values of k₋₁ and k₋₂ are found with less-purified protein preparations. The values of k₋₁ and k₋₂ satisfy conditions required by previous ¹⁸O-incorporation studies of H₂¹⁸O into the P_i moiety on ATP hydrolysis and suggest that the cleavage step does involve hydrolysis of ATP or formation of an adduct between ATP and water. The equilibrium constant for the cleavage step at the myosin active site is 9. If the cycle of events during muscle contraction is described by the model proposed by Lymn & Taylor (1971), the fact that there is only a small negative standard free-energy change for the cleavage step is advantageous for efficient chemical to mechanical energy exchange during muscle contraction.     

Conformational states of sarcoplasmic reticulum Ca2+-ATPase as studied by proteolytic cleavage

Summary Conformational states in sarcoplasmic reticulum Ca²⁺-ATPase have been examined by tryptic and chymotryptic cleavage. High affinity Ca²⁺ binding (E₁ state) exposes a peptide bond in the A fragment of the polypeptide chain to trypsin. Absence of Ca²⁺ (E₂ state) exposes bonds in the B fragment, which are protected by binding of Mg²⁺ or ATP. After phosphorylation from ATP the tryptic cleavage pattern depends on the predominant phosphoenzyme species present. ADP-sensitive E₁P and ADP-insensitive E₂P have cleavage patterns identical to those of unphosphorylated E₁ and E₂, respectively, indicating that two major conformational states are involved in Ca²⁺ translocation. The transition from E₁P to E₂P is inhibited by secondary tryptic splits in the A fragment, suggesting that parts of this fragment are of particular importance for the energy transduction process.The tryptic cleavage patterns of phosphorylated forms of detergent solubilized monomeric Ca²⁺-ATPase were similar to those of the membrane-bound enzyme, indicating that Ca²⁺ translocation depends mainly on structural changes within a single peptide chain. On the other hand, the protection of the second cleavage site as observed after vanadate binding to membranous Ca²⁺-ATPase could not be achieved in the soluble monomeric enzyme. Shielding of this peptide bond may therefore be due to protein-protein interactions in the semicrystalline state of the vanadate-bound Ca²⁺-ATPase in membranous form.     

DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis

The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R₂N₂ stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6–7 nucleotides) downstream of the asymmetric recognition sequence 5′-GCCGC-3′. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage.     

Analysis of linkage positions in saccharomyces cerevisiaed-mannans by the reductive-cleavage method

The positions of linkage in the d-mannans derived from Saccharomyces cerevisiae X2180 and its mutants, mnn1, mnn2, and mnn4, were established by perethylation and subsequent reductive cleavage with triethylsilane in the presence of boron trifluoride etherate (BF₃ · Et₂O) or trimethylsilyl trifluoromethanesulfonate. With the latter as the catalyst, all glycosidic carbon-oxygen bonds were cleaved, to produce a mixture of ethylated 1,5-anhydro-d-mannitol derivatives. With BF₃ · Et₂O as the catalyst, 2-, 3-, and 6-linked residues were incompletely cleaved, and residues linked at both O-2 and O-6 were not cleaved at all. It was concluded that reductive cleavage is an attractive method for determination of the structure of polysaccharides.     

A hingeless Fc fusion system for site-specific cleavage by IdeS

Fusion of proteins to the Fc region of IgG is widely used to express cellular receptors and other extracellular proteins, but cleavage of the fusion partner is sometimes required for downstream applications. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a protease with exquisite specificity for human IgG, and it can also cleave Fc-fusion proteins at a single site in the N-terminal region of the CH2 domain. However, the site of IdeS cleavage results in the disulfide-linked hinge region partitioning with the released protein, complicating downstream usage of the cleaved product. To tailor the Fc fragment for release of partner proteins by IdeS treatment, we investigated the effect of deleting regions of IgG-derived sequence that are upstream of the cleavage site. Elimination of the IgG-derived hinge sequence along with several residues of the CH2 domain had negligible effects on expression and purity of the fusion protein, while retaining efficient processing by IdeS. An optimal Fc fragment comprising residues 235–447 of the human IgG1 heavy chain sufficed for efficient production of fusion proteins and minimized the amount of residual Ig-derived sequence on the cleavage product following IdeS treatment. Pairing of this truncated Fc fragment with IdeS cleavage enables highly specific cleavage of Fc-fusion proteins, thus eliminating the need to engineer extraneous cleavage sequences. This system should be helpful for producing Fc-fusion proteins requiring downstream cleavage, particularly those that are sensitive to internal miscleavage if treated with alternative proteases.     

Stoichiometry of two plant glycine decarboxylase complexes and comparison with a cyanobacterial glycine cleavage system

The multienzyme glycine cleavage system (GCS) converts glycine and tetrahydrofolate to the one‐carbon compound 5,10‐methylenetetrahydrofolate, which is of vital importance for most if not all organisms. Photorespiring plant mitochondria contain very high levels of GCS proteins organised as a fragile glycine decarboxylase complex (GDC). The aim of this study is to provide mass spectrometry‐based stoichiometric data for the plant leaf GDC and examine whether complex formation could be a general property of the GCS in photosynthesizing organisms. The molar ratios of the leaf GDC component proteins are 1L₂‐4P₂‐8T‐26H and 1L₂‐4P₂‐8T‐20H for pea and Arabidopsis, respectively, as determined by mass spectrometry. The minimum mass of the plant leaf GDC ranges from 1550 to 1650 kDa, which is larger than previously assumed. The Arabidopsis GDC contains four times more of the isoforms GCS‐P1 and GCS‐L1 in comparison with GCS‐P2 and GCS‐L2, respectively, whereas the H‐isoproteins GCS‐H1 and GCS‐H3 are fully redundant as indicated by their about equal amounts. Isoform GCS‐H2 is not present in leaf mitochondria. In the cyanobacterium Synechocystis sp. PCC 6803, GCS proteins concentrations are low but above the complex formation threshold reported for pea leaf GDC. Indeed, formation of a cyanobacterial GDC from the individual recombinant GCS proteins in vitro could be demonstrated. Presence and metabolic significance of a Synechocystis GDC in vivo remain to be examined but could involve multimers of the GCS H‐protein that dynamically crosslink the three GCS enzyme proteins, facilitating glycine metabolism by the formation of multienzyme metabolic complexes. Data are available via ProteomeXchange with identifier PXD018211.     

Mechanism of an in vitro biosynthesis of thyroid hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine: I. Manifestation of the elimination of the alanine side chain of the N-terminal diiodotyrosine

It was previously shown that 3,5-diido-l-tyrosyl-3,5-diiodo-l-tyrosine, I₂Tyr-I₂Tyr, acts as a precursor in the in vitro synthesis of thyroid hormones, and a mechanism of syntheis was proposed.We investigated this pathway by incubations of I₂Tyr-I₂Tyr with microsomal solubilized thyroid proteins. I₂Tyr-T₂Tyr was doubly labeled: iodinated with ¹³¹I on the ring and tritiated either on the alanine side-chain of the N- or C-terminal diidotyrosine. It is shown that only the C-terminal alanine participates in the synthesis, the N-terminal alanine being eliminated.The result proved that I₂Tyr-I₂Tyr acts as precursor through a mechanism which is different from the one involving I₂Tyr. This mechanism consists of: Schiff base formation with pyridoxal; free radical formation and cyclization; peptide bond cleavage and removal of the pyridoxal · alanine complex.     

Mechanism of an in vitro biosynthesis of thyroid hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine: I. Manifestation of the elimination of the alanine side chain of the N-terminal diiodotyrosine

It was previously shown that 3,5-diido-l-tyrosyl-3,5-diiodo-l-tyrosine, I₂Tyr-I₂Tyr, acts as a precursor in the in vitro synthesis of thyroid hormones, and a mechanism of syntheis was proposed.We investigated this pathway by incubations of I₂Tyr-I₂Tyr with microsomal solubilized thyroid proteins. I₂Tyr-T₂Tyr was doubly labeled: iodinated with ¹³¹I on the ring and tritiated either on the alanine side-chain of the N- or C-terminal diidotyrosine. It is shown that only the C-terminal alanine participates in the synthesis, the N-terminal alanine being eliminated.The result proved that I₂Tyr-I₂Tyr acts as precursor through a mechanism which is different from the one involving I₂Tyr. This mechanism consists of: Schiff base formation with pyridoxal; free radical formation and cyclization; peptide bond cleavage and removal of the pyridoxal · alanine complex.     

Steric effects, solvent effects, and turnover in hydrolytic cleavage of peptides promoted by palladium(II) aqua complexes

 Dipeptides and tripeptides AcMet-aaH containing N-acetyl methionine, in which the group aaH is GlyH, AlaH, ValH, or Gly-GlyH, undergo hydrolytic cleavage of the Met-aaH peptide bond in the presence of the following complexes of palladium(II): cis-[Pd(en)(H₂O)₂]²⁺, cis-[Pd(tn)(H₂O)₂]²⁺, cis-[Pd(en)(CH₃OH)₂]²⁺, cis-[Pd(S,N-MetH)(H₂O)₂]²⁺, cis-[Pd(S,N-Met-GlyH)(H₂O)₂]²⁺, and cis-[Pd(S,N-Met-AlaH)(H₂O)₂]²⁺. These mononuclear complexes are precursors of binuclear palladium(II) complexes containing the substrates AcMet-aaH as bridging thioether ligands. The rate constant for cleavage is higher when the bidentate ligand in the precursor complex is ethylenediamine (which is completely displaced) than S,N-methionine (of which only the amino group is displaced), because the number of aqua ligands available for cleavage is greater in the former than in the latter case. The demonstrated dependence of the rate constant on the steric bulk (volume) of the leaving group, aaH, points the way toward achieving a degree of sequence selectivity in cleavage of peptide bonds by palladium(II) aqua complexes. One equivalent of cis-[Pd(en)(H₂O)₂]²⁺ cleaves as many as ten equivalents of AcMet-GlyH, but the rate constant decreases as the molar excess of the dipeptide over the catalyst increases. This demonstration of catalytic turnover points the way to our ultimate goal – artificial metallopeptidases. Received: 13 June 1997 / Accepted: 24 September 1997     

In planta cleavage of carotenoids by <Emphasis Type="Italic">Arabidopsis</Emphasis> carotenoid cleavage dioxygenase 4 in transgenic rice plants

A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C₄₀) cleavage products, a two-fold increase of β-ionone (C₁₃) and de novo generation of β-cyclocitral (C₁₀) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).     

Chemically and photochemically initiated DNA cleavage by an insulin-mimeticbisperoxovanadium complex

Chemically and photochemically induced cleavage of DNA by the insulin-mimetic compound NH₄[VO(O₂)₂-(1,10-phenanthroline)], bpV(phen), have been studied.⁵¹V NMR and absorption indicate that photoirradiation with low energy UV light of aqueous solutions containing bpV(phen) leads to the conversion of the compound to simple vanadates. Photoillumination of the compound in the presence of supercoiled pBR322 DNA results in cutting of the plasmid to produce nicked circular and linear DNA. Quantitative analysis of agarose gel data shows that bpV(phen) is a single strand nicking agent exhibiting sequence and/or base specificity.     

Differential regulation of apoptosis by caspase-mediated cleavage of phospholipase D isozymes

Phospholipase D (PLD) has been implicated in survival and anti-apoptosis, but the molecular mechanism by which it responds to apoptotic stimuli is poorly unknown. Here, we demonstrate that cleavage of PLD isozymes as specific substrates of caspase differentially regulates apoptosis. PLD1 is cleaved at one internal site (DDVD₅₄₅S) and PLD2 is cleaved at two or three sites (PTGD₁₃ELD₁₆S and DEVD₂₈T) in the front of N-terminus. Cleavage of PLD was endogenously detected in post-mortem Alzheimer brain together with activated caspase-3, suggesting the physiological relevance. The cleavage of PLD1 but not PLD2 might act as an inactivating process since PLD1 but not PLD2 activity is significantly decreased during apoptosis, suggesting that differential cleavage of PLD isozymes could affect its enzymatic activity. Moreover, caspase-resistant mutant of PLD1 showed more potent anti-apoptotic capacity than that of wild type PLD1, whereas PLD2 maintained anti-apoptotic potency in spite of its cleavage during apoptosis. Moreover, PLD2 showed more potent anti-apoptotic effect than that of PLD1 in overexpression and knockdown experiments, suggesting that difference in anti-apoptotic potency between PLD1 and PLD2 might be due to its intrinsic protein property. Taken together, our results demonstrate that differential cleavage pattern of PLD isozymes by caspase might affect its enzymatic activity and anti-apoptotic function.     

Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

We have characterized a novel Zn²⁺-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn²⁺. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn²⁺-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes.