Dimer Exchange Research Articles

Nucleosome flipping drives kinetic proofreading and processivity by SWR1

The yeast SWR1 complex catalyses the exchange of histone H2A–H2B dimers in nucleosomes, with Htz1–H2B dimers1, 2–3. Here we used single-molecule analysis to demonstrate two-step double exchange of the two H2A–H2B dimers in a canonical yeast nucleosome with Htz1–H2B dimers, and showed that double exchange can be processive without release of the nucleosome from the SWR1 complex. Further analysis showed that bound nucleosomes flip between two states, with each presenting a different face, and hence histone dimer, to SWR1. The bound dwell time is longer when an H2A–H2B dimer is presented for exchange than when presented with an Htz1–H2B dimer. A hexasome intermediate in the reaction is bound to the SWR1 complex in a single orientation with the ‘empty’ site presented for dimer insertion. Cryo-electron microscopy analysis revealed different populations of complexes showing nucleosomes caught ‘flipping’ between different conformations without release, each placing a different dimer into position for exchange, with the Swc2 subunit having a key role in this process. Together, the data reveal a processive mechanism for double dimer exchange that explains how SWR1 can ‘proofread’ the dimer identities within nucleosomes.

Structure of the human TIP60 complex

Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a ‘pincer-like’ conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5–nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C.

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a 'pincer-like' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

Multiple strongly coupled antiferromagnetic spin S = 1/2 dimers in liroconite Cu2Al(As,P)O4(OH)4·4H2O

Abstract We report on the magneto-structural properties of the rare copper aluminum hydroxo-arsenate mineral liroconite with chemical composition Cu2AlAs1−x P x O4(OH)4·4H2O (x ≈ 0.2). In order to characterize the natural mineral sample chemical analyses, X-ray single crystal and powder diffraction, heat capacity and crystal water desorption, anisotropic thermal expansion and Raman scattering and magnetic susceptibility investigations have been carried out. The magnetic properties are well described by two discrete oxygen bridged Cu2+ spin S = 1/2 dimers with antiferromagnetic spin exchange ranging between −320 K and −136 K, depending on to which group-15 five-valent cation, As5+ or P5+, the dimer bridging oxygen atoms coordinate to. Accordingly the temperature dependence of the magnetic susceptibilities can be well fitted to a sum of two Bleaney–Bowers type spin S = 1/2 dimer susceptibilities suggesting that the dimers show negligible mixed coordination to (AsO4)3−/(PO4)3− tetrahedra. DFT + U calculation confirm the ratio of the spin exchange parameters of the (AsO4)3− or (PO4)3− coordinated Cu2+ – Cu2+ dimers. Inter dimer spin exchange is about two orders of magnitude smaller than intra dimer exchange.

Tracking the evolution from isolated dimers to many-body entanglement in NaLuxYb1−xSe2

We synthesize homogeneous compositions of NaLu$_x$Yb$_{1-x}$Se$_2$, connecting non-magnetic NaLuSe$_2$ to the triangular lattice spin liquid candidate NaYbSe$_2$. Thermal and magnetic properties are studied as the system evolves from one with dilute magnetic defects to one of a dense magnetic lattice. The field and temperature dependent heat capacity show the carriers of entropy crossover from isolated magnetic ions to a correlated lattice borne from spin dimers. For the dilute system we estimate the single ion anisotropy $(g_\perp/g_\parallel =3.13)$ and also the dimer exchange couplings $J_\parallel(=5.4$~K) and $J_\perp(=9.6$~K), in order to draw comparison to the half-doped and full magnetic compounds.

Motor usage imprints microtubule stability along the shaft

Tubulin dimers assemble into dynamic microtubules, which are used by molecular motors as tracks for intracellular transport. Organization and dynamics of the microtubule network are commonly thought to be regulated at the polymer ends, where tubulin dimers can be added or removed. Here, we show that molecular motors running on microtubules cause exchange of dimers along the shaft invitro and in cells. These sites of dimer exchange act as rescue sites where depolymerizing microtubules stop shrinking and start re-growing. Consequently, the average length of microtubules increases depending on how frequently they are used as motor tracks. An increase of motor activity densifies the cellular microtubule network and enhances cell polarity. Running motors leave marks in the shaft, serving as traces of microtubule usage to organize the polarity landscape of the cell.

D 5 off-centering induced ferroelectric and magnetoelectric correlations in trirutile-Fe2TeO6

We present the rare existence of d5 off-centering induced weak ferroelectric polarization and demonstrate its correlation with observed magnetoelectric (ME) properties in the G type (TN ∼ 210 K) antiferromagnet Fe2TeO6 (FTO) compound. The origin of ferroelectricity (FE) is associated with both lattice and asymmetric electron density (ED) distribution around the ion cores. ME coupling is observed in magnetic field-dependent polarization, ME voltage, and magnetostrain measurements. Short-range magnetic ordering due to intrabilayer dimeric exchange coupling via the double oxygen bridged Fe–O1–Fe pathway is proposed to play a dominating role to exhibit the negative nonlinear magnetic field-dependent ME behavior at 300 K. Interbilayer exchange via Fe–O2–Fe pathways dominantly determines the hysteretic nonlinear magnetic field-dependent ME response below TN. The observed nonlinear ME coupling signifies magnetoelasticity as manifested in the temperature and magnetic field-dependent strain measurement. Hence, the rare existence of FE and magnetoelectric coupling by d5 ion is presented in FTO.

Thermodynamics of General Heisenberg Spin Tetramers Composed of Coupled Quantum Dimers

Advances in quantum computing technology have been made in recent years due to the evolution of spin clusters. Recent studies have tended towards spin cluster subgeometries to understand more complex structures better. These molecular magnets provide a multitude of phenomena via exchange interactions that allow for advancements in spintronics and other magnetic system applications due to the possibility of increasing speed, data storage, memory, and stability of quantum computing systems. Using the Heisenberg spin–spin exchange Hamiltonian and exact diagonalization, we examine the evolution of quantum energy levels and thermodynamic properties for various spin configurations and exchange interactions. The XXYY quantum spin tetramer considered in this study consists of two coupled dimers with exchange interactions α1J and α1′J and a dimer–dimer exchange interaction α2J. By varying spin values and interaction strengths, we determine the exact energy eigenstates that are used to determine closed-form analytic solutions for the heat capacity and magnetic susceptibility of the system and further analyze the evolution of the properties of the system based on the parameter values chosen. Furthermore, this study shows that the Schottky anomaly shifts towards zero as the ground-state of the system approaches a quantum phase transition between spin states. Additionally, we investigate the development of phase transitions produced by the convergence of the Schottky anomaly with both variable exchange interactions and external magnetic field.

The intrinsic stability of H2B-ubiquitylated nucleosomes and their in vitro assembly/disassembly by histone chaperone NAP1

BackgroundApart the gene-regulatory functions as docking sites for histone ‘readers’, some histone modifications could directly affect nucleosome structure. The H2BK34-ubiquitylation deposited by MOF-MSL complex, increases nucleosome dynamics in vitro and promotes donation of one H2A/H2B dimer to histone acceptors. MethodsWe evaluated temperature-depended stability of H2BK34-ubiquitylated nucleosomes under ‘physiological’ ionic conditions in the presence or absence of histone acceptor, and examined assembly and disassembly of ubiquitylated nucleosomes in vitro by recombinant mouse NAP1. ResultsH2BK34ub modification is sufficient to promote selective eviction of only one H2A/H2B dimer independently of histone-binding agents. Despite the robust H2A/H2B dimer-displacement effect of mNAP1 with the H2BK34ub (but not unmodified) nucleosomes, NAP1 could assemble symmetrically- or asymmetrically ubiquitylated nucleosomes under ‘physiological’ conditions in vitro. Conclusions and general significanceThe increased mobility of one nucleosomal H2A/H2B dimer is an intrinsic nucleosome destabilizing property of H2BK34 ubiquitylation that has the intranucleosome bases. The ability of NAP to reasonably efficiently assemble H2BK34-ubiquitylated nucleosomes supposes a potential mechanism for deposition/distribution of H2BK34ub mark in the MOF-MSL independent manner (for example, during histone dimer exchange upon transcription elongation).

A mixture of three engineered phosphotriesterases enables rapid detoxification of the entire spectrum of known threat nerve agents

Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase (Bd-PTE) that are highly stable and nearly sequence identical. This mixture enables effective detoxification of a broad spectrum of known threat agents-GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), VX and Russian-VX. The potential for dimer dissociation and exchange that could inactivate Bd-PTE has minimal impact, and the three enzyme variants are as active in a mixture as they are individually. To our knowledge, this engineered enzyme 'cocktail' comprises the first solution for enzymatic detoxification of the entire range of threat nerve agents.

Exclusivity and Compensation in NFκB Dimer Distributions and IκB Inhibition.

The NFκB transcription factor family members RelA, p50, and cRel form homo- and heterodimers that are inhibited by IκBα, IκBβ, and IκBε. These NFκB family members have diverse biological functions, and their expression profiles differ, leading to different concentrations in different tissue types. Here we present definitive biophysical measurements of the NFκB dimer affinities and inhibitor affinities to better understand dimer exchange and how the presence of inhibitors may alter the equilibrium concentrations of NFκB dimers in the cellular context. Fluorescence anisotropy binding experiments were performed at low concentrations to mimic intracellular concentrations. We report binding affinities much stronger than those that had been previously reported by non-equilibrium gel shift and analytical ultracentrifugation assays. The results reveal a wide range of NFκB dimer affinities and a strong preference of each IκB for a small subset of NFκB dimers. Once the preferred IκB is bound, dimer exchange no longer occurs over a period of days. A mathematical model of the cellular distribution of these canonical NFκB transcription factors based on the revised binding affinities recapitulates intracellular observations and provides simple, precise explanations for observed cellular phenomena.

Transient Kinetic Analysis of SWR1C-Catalyzed H2A.Z Deposition Unravels the Impact of Nucleosome Dynamics and the Asymmetry of Histone Exchange

SUMMARYThe SWR1C chromatin remodeling enzyme catalyzes ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes such as transcription and DNA repair. Here, we investigate the transient kinetic mechanism of the histone exchange reaction, employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady-state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATP-dependent replacement of nucleosomal dimers, promoted by localized DNA unwrapping. Taken together, our transient kinetic studies identify intermediates and provide crucial insights into the SWR1C-catalyzed dimer exchange reaction and shed light on how the mechanics of H2A.Z deposition might contribute to transcriptional regulation in vivo.

DRES-17. BCL3 IS A 19q PASSENGER THAT PREDICTS RESPONSE TO ALKYLATING CHEMOTHERAPY IN GBM

Despite the universal use of temozolomide (TMZ) in GBM, many patients experience minimal benefit. Moreover, TMZ can be deleterious due to its mutagenic nature. These findings underline the importance of predicting which patients will benefit from TMZ. DATA: Bcl-3 was identified as being involved in cytotoxicity by TMZ due to its unique interaction with NF-κB residues modified following treatment. Up- and down-regulation of Bcl-3 in patient-derived glioma stem-like cells (GSCs) demonstrates that Bcl-3 blocks cytotoxicity by TMZ in vitro and in an intracranial PDX. Analysis of multiple public GBM datasets demonstrates that BCL3 mRNA expression is a marker of outcome, a finding confirmed on multivariate analysis incorporating MGMT promoter methylation, age and IDH mutation among other variables. Most notably, separation of patients in TCGA by treatment modality demonstrates that BCL3 is only informative in patients who received alkylating chemotherapy not untreated patients or those receiving ionizing radiation (IR) alone, a finding significant on multivariate analysis and validated in REMBRANDT. To examine Bcl-3 protein, TMAs were constructed incorporating 86 GBMs. Bcl-3 immunohistochemistry graded on a 4-tier system and dichotomized significantly separates patients into survival groups (P<0.0001; HR, 5.418; 95%CI, 2.747–9.647). In GBM, Bcl-3 mRNA and protein level is regulated by gene copy number determined in public datasets and in TMAs by FISH. CNAs of BCL3 occur due to changes directed to a broad region of 19q13, an area deleted in 20–40% of all types of glioma. Mechanistically, Bcl-3 promotes resistance by inducing mesenchymal differentiation via promoter-specific NF-κB dimer exchange, shown in GSCs and confirmed in TCGA GBM. BCL3 is an independent predictor of response to TMZ in GBM whose expression is regulated by CNA targeted to 19q13 that inadvertently affect BCL3. Bcl-3 expression level represents a cell-intrinsic modulator of NF-κB activity that promotes mesenchymal differentiation and resistance to therapy.

The INO80 remodeller in transcription, replication and repair.

The accessibility of eukaryotic genomes to the action of enzymes involved in transcription, replication and repair is maintained despite the organization of DNA into nucleosomes. This access is often regulated by the action of ATP-dependent nucleosome remodellers. The INO80 class of nucleosome remodellers has unique structural features and it is implicated in a diverse array of functions, including transcriptional regulation, DNA replication and DNA repair. Underlying these diverse functions is the catalytic activity of the main ATPase subunit, which in the context of a multisubunit complex can shift nucleosomes and carry out histone dimer exchange. In vitro studies showed that INO80 promotes replication fork progression on a chromatin template, while in vivo it was shown to facilitate replication fork restart after stalling and to help evict RNA polymerase II at transcribed genes following the collision of a replication fork with transcription. More recent work in yeast implicates INO80 in the general eviction and degradation of nucleosomes following high doses of oxidative DNA damage. Beyond these replication and repair functions, INO80 was shown to repress inappropriate transcription at promoters in the opposite direction to the coding sequence. Here we discuss the ways in which INO80's diverse functions help maintain genome integrity.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD.

The Escherichia coli SOS response, an induced DNA damage response pathway, confers survival on bacterial cells by providing accurate repair mechanisms as well as the potentially mutagenic pathway translesion synthesis (TLS). The umuD gene products are upregulated after DNA damage and play roles in both nonmutagenic and mutagenic aspects of the SOS response. Full-length UmuD is expressed as a homodimer of 139-amino-acid subunits, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The cleavage product UmuD' and UmuC form the Y-family polymerase DNA Pol V (UmuD'2C) capable of performing TLS. UmuD and UmuD' exist as homodimers, but their subunits can readily exchange to form UmuDD' heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway of UmuD'. The recognition sequence for ClpXP protease is located within the first 24 amino acids of full-length UmuD, and the partner of full-length UmuD, whether UmuD or UmuD', is degraded by ClpXP. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange of a number of fluorescently labeled single-cysteine UmuD variants as detected by Förster resonance energy transfer. Labeling sites near the dimer interface correlate with increased rates of exchange, indicating that weakening the dimer interface facilitates exchange, whereas labeling sites on the exterior decrease the rate of exchange. In most but not all cases, homodimer and heterodimer exchange exhibit similar rates, indicating that somewhat different molecular surfaces mediate homodimer exchange and heterodimer formation.

INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers

ATP-dependent chromatin remodellers modulate nucleosome dynamics by mobilizing or disassembling nucleosomes, as well as altering nucleosome composition. These chromatin remodellers generally function by translocating along nucleosomal DNA at the H3–H4 interface of nucleosomes. Here we show that, unlike other remodellers, INO80 translocates along DNA at the H2A–H2B interface of nucleosomes and persistently displaces DNA from the surface of H2A–H2B. DNA translocation and DNA torsional strain created near the entry site of nucleosomes by INO80 promotes both the mobilization of nucleosomes and the selective exchange of H2A.Z–H2B dimers out of nucleosomes and replacement by H2A–H2B dimers without any additional histone chaperones. We find that INO80 translocates and mobilizes H2A.Z-containing nucleosomes more efficiently than those containing H2A, partially accounting for the preference of INO80 to replace H2A.Z with H2A. Our data suggest that INO80 has a mechanism for dimer exchange that is distinct from other chromatin remodellers including its paralogue SWR1.

Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD

The SOS response, an induced DNA damage response pathway in Escherichia coli, is temporally separated into two stages. The initial stage affords an opportunity for the cell to utilize accurate repair mechanisms such as homologous recombination and nucleotide excision repair. During the later stage, cells employ a potentially mutagenic pathway termed translesion synthesis (TLS) to tolerate damage and promote survival. The umuD gene products are upregulated during the SOS response and play a role in both early and late stages. Full‐length UmuD is expressed as a homodimer of 139‐amino‐acid subunits which eventually cleave their N‐terminal 24 amino acids to form UmuD′. The cleavage product, UmuD′, together with UmuC, form the Y‐family polymerase DNA Pol V (UmuD′2C) capable of performing TLS. UmuD and UmuD′ exist as homodimers, but their subunits can readily exchange to form UmuDD′ heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway for the pro‐mutagenic UmuD′. Full‐length UmuD is targeted for degradation by the ClpXP protease due to a recognition sequence located on the N‐terminal arm of UmuD. Formation of UmuD′ via cleavage removes this recognition sequence, making UmuD′ invisible to ClpX. Thus, UmuD in the context of heterodimer acts as a delivery factor for UmuD′. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange for a number of single‐cysteine UmuD variants. By conjugating fluorescent molecules at different positions on the protein we were able to observe their effect on exchange kinetics. Interestingly, the most dramatic differential effects on exchange were observed at two adjacent positions in the peptide backbone. These findings provide insight into the molecular surfaces responsible for mediating exchange.Support or Funding InformationSupported by NSF CAREER MCB‐0845033 and American Cancer Society RSG‐12‐161‐01‐DMC.

Response to Comment on "A histone acetylation switch regulates H2A.Z deposition by the SWR-C remodeling enzyme".

Wang et al report a failure to reproduce our biochemical observation that the INO80C and SWR1C/SWR1/SWR-C chromatin remodeling enzymes catalyze replacement of nucleosomal H2A.Z with H2A when the substrate contains H3-K56Q. They point to technical problems with our dimer exchange assay. In response, we have recapitulated our findings using a mobility shift assay that was developed and employed by Wang and colleagues.

Evidence for an essential role of intradimer interaction in catalytic function of carnosine dipeptidase II using electrospray-ionization mass spectrometry

Carnosine dipeptidase II (CN2/CNDP2) is an M20 family metallopeptidase that hydrolyses various dipeptides including β-alanyl-L-histidine (carnosine). Crystallographic analysis showed that CN2 monomer is composed of one catalytic and one dimerization domains, and likely to form homodimer. In this crystal, H228 residue of the dimerization domain interacts with the substrate analogue bestatin on the active site of the dimer counterpart, indicating that H228 is involved in enzymatic reaction. In the present study, the role of intradimer interaction of CN2 in its catalytic activity was investigated using electrospray-ionization time-of-flight mass spectrometry (ESI-TOF MS). First, a dimer interface mutant I319K was prepared and shown to be present as a folded monomer in solution as examined by using ESI-TOF MS. Since the mutant was inactive, it was suggested that dimer formation is essential to its enzymatic activity. Next, we prepared H228A and D132A mutant proteins with different N-terminal extended sequences, which enabled us to monitor dimer exchange reaction by ESI-TOF MS. The D132A mutant is a metal ligand mutant and also inactive. But the activity was partially recovered time-dependently when H228A and D132A mutant proteins were incubated together. In parallel, H228A/D132A heterodimer was formed as detected by ESI-TOF MS, indicating that interaction of a catalytic center with H228 residue of the other subunit is essential to the enzymatic reaction. These results provide evidence showing that intradimer interaction of H228 with the reaction center of the dimer counterpart is essential to the enzymatic activity of CN2.