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Lung adenocarcinoma (LUAD) is the most challenging neoplasm to treat in clinical practice. Ankyrin repeat domain 49 protein (ANKRD49) is highly expressed in several carcinomas; however, its pattern of expression and role in LUAD are not known. Tissue microarrays, immunohistochemistry, χ

Directional cell migration involves signaling cascades that stimulate actin assembly at the leading edge, and additional pathways must inhibit actin polymerization at the rear. During neuroblast migration in Caenorhabditis elegans, the transmembrane protein MIG-13/Lrp12 acts through the Arp2/3 nucleation-promoting factors WAVE and WASP to guide the anterior migration. Here we show that a tyrosine kinase, SRC-1, directly phosphorylates MIG-13 and promotes its activity on actin assembly at the leading edge. In GFP knockin animals, SRC-1 and MIG-13 distribute along the entire plasma membrane of migrating cells. We reveal that a receptor-like tyrosine phosphatase, PTP-3, maintains the F-actin polarity during neuroblast migration. Recombinant PTP-3 dephosphorylates SRC-1–dependent MIG-13 phosphorylation in vitro. Importantly, the endogenous PTP-3 accumulates at the rear of the migrating neuroblast, and its extracellular domain is essential for directional cell migration. We provide evidence that the asymmetrically localized tyrosine phosphatase PTP-3 spatially restricts MIG-13/Lrp12 receptor activity in migrating cells.

The activity of 5′-adenosine monophosphate-activated protein kinase (AMPK) is inversely correlated with the cellular availability of glucose. When glucose levels are low, the glycolytic enzyme aldolase is not bound to fructose-1,6-bisphosphate (FBP) and, instead, signals to activate lysosomal AMPK. Here, we show that blocking FBP binding to aldolase with the small molecule aldometanib selectively activates the lysosomal pool of AMPK and has beneficial metabolic effects in rodents. We identify aldometanib in a screen for aldolase inhibitors and show that it prevents FBP from binding to v-ATPase-associated aldolase and activates lysosomal AMPK, thereby mimicking a cellular state of glucose starvation. In male mice, aldometanib elicits an insulin-independent glucose-lowering effect, without causing hypoglycaemia. Aldometanib also alleviates fatty liver and nonalcoholic steatohepatitis in obese male rodents. Moreover, aldometanib extends lifespan and healthspan in both Caenorhabditis elegans and mice. Taken together, aldometanib mimics and adopts the lysosomal AMPK activation pathway associated with glucose starvation to exert physiological roles, and might have potential as a therapeutic for metabolic disorders in humans.

The SAD (synapses of amphids defective) kinases, including SAD-A and SAD-B, play important roles in the regulation of neuronal development, cell cycle, and energy metabolism. Our recent study of mouse SAD-A identified a unique autoinhibitory sequence (AIS), which binds at the junction of the kinase domain (KD) and the ubiquitin-associated (UBA) domain and exerts autoregulation in cooperation with UBA. Here, we report the crystal structure of the mouse SAD-B C-terminal fragment including the AIS and the kinase-associated domain 1 (KA1) at 2.8 Å resolution. The KA1 domain is structurally conserved, while the isolated AIS sequence is highly flexible and solvent-accessible. Our biochemical studies indicated that the SAD-B AIS exerts the same autoinhibitory role as that in SAD-A. We believe that the flexible isolated AIS sequence is readily available for interaction with KD-UBA and thus inhibits SAD-B activity.

Currently, liver cancer is the sixth most prevalent cancer and the third most common cause of cancer-related death. However, effective chemotherapeutic drugs with low drug resistance and few side-effects for the clinical treatment of liver cancer are lacking. Therefore, the search for novel drugs to compensate for the defects of existing drugs is urgently needed. Herein, we successfully screened an extract named from Stellera chamaejasme L. (SCL), a historically confimed antitumor plant, through a novel extraction platform. In the present study, we firstly screened the anticancer effect of ESC by the sulforhodamine B (SRB) cell proliferation assay in a wide range of malignant cell lines, including A549, NCI-H157, NCI-H460, SK-HEP-1 and HepG2. With the highest inhibitory rate in hepatocarcinoma cells, we further identified the tumor-suppressive efficacy and the safety of ESC in an H22 hepatocarcinoma xenograft model in vivo. In a mechanistic study, flow cytometry and western blot analysis were performed to evaluate the effects of ESC on the induction of cell apoptosis, intervention of cell cycle distribution and its influence on key G2/M-phase regulators. The results showed that ESC significantly inhibited the cell growth of liver cancer cell lines. Accordingly, the tumor inhibition rate was also increased following ESC administration with little systemic toxicity in H22-transplanted mice. Mechanistically, ESC caused obvious G2/M-phase arrest in both the SK-HEP-1 and HepG2 cell lines without cell apoptosis. Furthermore, cyclin B1 was downregulated, while the phosphorylation level of CDK1 was increased in response to ESC treatment. All these data confirmed that ESC possesses potent anti-proliferative efficacy for hepatocarcinoma through the induction of cyclin-mediated cell cycle arrest. Thus, ESC is a promising candidate for hepatocarcinoma treatment in the future.

The Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN), a heterodimer composed of a catalytic subunit A and an essential regulatory subunit B, plays critical functions in various cellular processes such as cardiac hypertrophy and T cell activation. It is the target of the most widely used immunosuppressants for transplantation, tacrolimus (FK506) and cyclosporin A. However, the structure of a large part of the CNA regulatory region remains to be determined, and there has been considerable debate concerning the regulation of CN activity. Here, we report the crystal structure of full-length CN (β isoform), which revealed a novel autoinhibitory segment (AIS) in addition to the well-known autoinhibitory domain (AID). The AIS nestles in a hydrophobic intersubunit groove, which overlaps the recognition site for substrates and immunosuppressant-immunophilin complexes. Indeed, disruption of this AIS interaction results in partial stimulation of CN activity. More importantly, our biochemical studies demonstrate that calmodulin does not remove AID from the active site, but only regulates the orientation of AID with respect to the catalytic core, causing incomplete activation of CN. Our findings challenge the current model for CN activation, and provide a better understanding of molecular mechanisms of CN activity regulation.

Bistability (coexistence of two stable steady states in a dynamical system) is a key mechanism of cellular decision-making and has been observed in many biochemical reaction networks such as mitogen-activated protein kinase (MAPK) signaling pathways. Theoretical studies have shown that bistability can arise in a single two-site MAPK phosphorylation and dephosphorylation cycle. However, the bistable behavior mostly relies on the kinetic mechanisms and parameters of this two-site modification. In exploring the system-level properties of MAPK regulation, most models to date focus on two limiting reaction regimes, distributive and processive, and are characterized by high levels of parametric uncertainty. Here, we developed a combined kinetic method which applies a continuous spectrophotometric enzyme-coupled assay incorporated with the viscosity approach, to perform detailed kinetic analyses of p38α MAPK dual phosphorylation by MKK6. Almost all kinetic rate constants for the first and second phosphorylation steps in p38α activation have been quantitatively determined, supporting that the phosphorylation occurs randomly in the first step, albeit preferring the tyrosine residue. The release rates of monophosphorylated p38α from MKK6, either as the product in the first modification or as the substrate in the second step, were comparable to the respective adjacent phosphoryl transfer steps. These results indicated that dual phosphorylation of p38α by MKK6 involves a random, partially processive mechanism. Based on the experimentally determined models and parameters, dynamics of the p38α-MKK6-MKP5 system were explored, demonstrating for the first time that bistability can arise with this model at biologically feasible parameter values. ENZYMES: p38α (EC 2.7.11.24); MKK6 (EC 2.7.12.2).

Brassinosteroids (BRs) are a class of essential polyhydroxylated steroid hormones regulating plant growth and development, and their responses to environmental cues (Clouse, 2011; Yang et al., 2011). BRs are perceived at the plasma membrane by a receptor complex, which consists of two leucine-rich receptor-like kinases (LRR-RLKs), BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). When activated by BRs, the receptor complex initiates a cascade of multiple kinases and phosphatases, ultimately leading to the dephosphorylation and activation of a family of closely related transcription factors, including BRI1-EMS SUPPRESSOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1), to regulate the expression of thousands of target genes (Guo et al., 2013).

The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are down-regulated by the MAPK phosphatases (MKPs). Catalytic activity of the MKPs is controlled both by their ability to recognize selective MAPKs and by allosteric activation upon binding to MAPK substrates. Here, we use a combination of experimental and computational techniques to elucidate the molecular mechanism for the ERK2-induced MKP3 activation. Mutational and kinetic study shows that the 334FNFM337 motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and responsible for ERK2-mediated MKP3 activation. The long-term molecular dynamics (MD) simulations further reveal a complete dynamic process, in which the catalytic domain of MKP3 gradually changes to a conformation that resembles an active MKP catalytic domain over the timescale of the simulation, providing a direct time-dependent observation of allosteric signal transmission in ERK2 induced MKP3 activation.

Sucrose non-fermenting (Snf1)-related kinase (SNRK) is a novel member of the AMP-activated protein kinase (AMPK) family and is involved in many metabolic processes. Here we report the crystal structure of an N-terminal SNRK fragment containing kinase and adjacent ubiquitin-associated (UBA) domains. This structure shows that the UBA domain binds between the N- and C-lobes of the kinase domain. The mode of UBA binding in SNRK largely resembles that in AMPK and brain specific kinase (BRSK), however, unique interactions play vital roles in stabilizing the KD-UBA interface of SNRK. We further propose a potential role of the UBA domain in the regulation of SNRK kinase activity. This study provides new insights into the structural diversities of the AMPK kinase family.

To study the immunologic function of dendritic cells (DCs) cultured in two kinds of hepatoma cell line's supernatant and the enhancing effects of carboxymethylpachymaran (CMP) on DCs.DCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines (HepG2 and HepG2.2.15) were collected for condition medium (CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium, which was 3:1. CMP was dissolved in incomplete RPMI-1640 medium. Experimental groups were divided according to the culture medium, either CM or with CMP in it. DCs subsets CD83, CD86, CD1a, and d-related human leukocyte antigens (HLA-DR) were analyzed by flow cytometry. The proliferation ability of allogeneic T cells in mixed lymphocyte reaction (MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. IL-12p70, interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) were detected by enzyme-linked immunosorbent assay analysis.The proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group (P0.01). Compared with the normal group, groups treated with CMP showed a higher expression level of DCs subsets, lymphocyte reproductive activity, as well as IL-12 and IFN-γ secretion levels. Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γ secretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group (P0.05). Compared with the normal group, the expression level of NF-κB in DCs nuclear was higher in CMP groups but lower in two CM groups (P0.05). After CMP was added, the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added (P0.05). However, there was no significant difference between the two CM groups (P0.05).Two kinds of hepatoma cell line's supernatant can inhibit the immunologic function of DCs. This suppressive effect may be related to the inhibition of NF-κB/Rel pathway. CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.

The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner.

MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38α in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38α is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38α can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38α substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38α but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38α has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.

Protein kinase activation, via autophosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation-dependent signaling cascades. Despite the prevalence of this reaction and its importance in biological regulation, the molecular mechanisms of autophosphorylation are poorly understood. In this study, we developed a kinetic approach to distinguish quantitatively between cis- and trans-pathways in an autocatalytic reaction. Using this method, we have undertaken a detailed kinetic analysis for the autoactivation mechanism of p21-activated protein kinase 2 (PAK2). PAK2 is regulated in vivo and in vitro by small GTP-binding proteins, Cdc42 and Rac. Full activation of PAK2 requires autophosphorylation of the conserved threonine, Thr402, in the activation loop of its catalytic kinase domain. Analyses of the time courses of substrate reaction during PAK2 autoactivation suggest that autophosphorylation of Thr402 in PAK2 obeys a two-step mechanism of cis initiation, followed by trans amplification. The unphosphorylated PAK2 undergoes an intramolecular (cis) autophosphorylation on Thr402 to produce phosphorylated PAK2, and this newly formed active PAK2 then phosphorylates other PAK2 molecules at Thr402 in an intermolecular (trans) manner. Based on the kinetic equation derived, all microscopic kinetic constants for the cis and trans autophosphorylation have been estimated quantitatively. The advantage of the new method is not only its usefulness in the study of fast activation reactions, but its convenience in the study of substrate effects on modification reaction. It would be particularly useful when the regulatory mechanism of the autophosphorylation reaction toward certain enzymes is being assessed.

In the present study, the enzymatic mechanism of ERK2 is re-examined by a combination of steady-state kinetic studies in the absence and presence of viscosogenic agents. Kinetic studies carried out in various concentrations of sucrose revealed that both kcat and kcat/Km for either ATP or EtsΔ138 were highlysensitive to solvent viscosity, suggesting that the rapid equilibrium assumption is not valid for the phosphorylation of protein substrate by ERK2. Furthermore, the kinetic analysis with the minimal random Bi Bi reaction mechanism is shown to be inconsistent with the principle of the detailed balance. This inconsistent calculation strongly suggests that there is isomerization of the enzyme-substrate ternary complex. The viscosity-dependent steady-state kinetic data are combined to establish a kinetic mechanism for the ERK2-catalyzed reaction that predicts initial reaction velocities under varying concentrations of ATP and substrate. These results complement previous structure-function studies of mitogen-activated protein kinases and provide important insight for mechanistic interpretation of the kinase functions.

Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction. Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function., The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs). Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction.

Members of the MAPK phosphatase (MKP) protein family play critical roles in immune responses through differential regulation of MAPK activation. In this study, we show that MKP7, also known as dual-specificity phosphatase 16, was required for CD4+ T cell responses in vivo. Mkp7−/− CD4+ T cells exhibited enhanced ERK and JNK activation, and produced increased amount of IL-2 compared with Mkp7+/+ cells upon activation. Mkp7−/− CD4+ T cells were selectively defective in Th17 differentiation in vitro, which was rescued by blocking IL-2 or inhibition of ERK activation. Furthermore, mice carrying Mkp7−/− T cells were deficient in generation of Th17 and T follicular helper cells in vivo, and were resistant to autoimmune experimental encephalomyelitis. Our results thus demonstrate an essential role of MKP7 in effector T cell function.

The present study investigates the molecular details of how arsenic trioxide inhibits preadipocyte differentiation and examines the role of Akt/PKB in regulation of differentiation and apoptosis. Continual exposure of arsenic trioxide, at the clinic achievable dosage that does not induce apoptosis, suppressed 3T3-L1 cell differentiation into fat cells by inhibiting the expression of PPARgamma and C/EBPalpha and disrupting the interaction between PPARgamma and RXRalpha, which determines the programming of the adipogenic genes. Interestingly, if we treated the cells for 12 or 24 h and then withdrew arsenic trioxide, the cells were able to differentiate to the comparable levels of untreated cells as assayed by the activity of GAPDH, the biochemical marker of preadipocyte differentiation. Long term treatment blocked the differentiation and the activity of GAPDH could not recover to the comparable levels of untreated cells. Continual exposure of arsenic trioxide caused accumulation in G2/M phase and the accumulation of p21. We found that arsenic trioxide induced the expression and the phosphorylation of Akt/PKB and it inhibited the interaction between Akt/PKB and PPARgamma . Akt/PKB inhibitor appears to block the arsenic trioxide suppression of differentiation. Our results suggested that Akt/PKB may play a role in suppression of apoptosis and negatively regulate preadipocyte differentiation.

arising from B. Xiao et al. , 230–233 (2011)10.1038/nature09932 The AMP-activated protein kinase (AMPK), an αβγ heterotrimeric enzyme, has a central role in regulating cellular metabolism and energy homeostasis1. The α-subunit of AMPK possesses the catalytic kinase domain, followed by a regulatory region comprising the autoinhibitory domain (AID) and α-linker2,3. Structural and biochemical studies suggested that AID is central to mammalian AMPK regulation4; however, this notion has been challenged recently by Xiao et al. on the basis of their active AMPK structure (Protein Data Bank accession 2Y94)5. On close inspection, however, we found that the α-subunit regulatory region was incorrectly built in their model, and our rebuilt model suggests a universal occurrence of the AID domain in AMPKs; we have also identified a novel regulatory motif that is essential for AMPK regulation.

The p21-activated kinases (PAKs) play an important role in diverse cellular processes. PAK2 is activated by autophosphorylation upon binding of small G proteins such as Cdc42 and Rac in the GTP-bound state. However, the mechanism of PAK2 autophosphorylation in vitro is unclear. In the present study, the kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the autoactivation of PAK2. On the basis of the kinetic equation of the substrate reaction during the autophosphorylation of PAK2, the activation rate constants for the free enzyme and enzyme-substrate complex have been determined. The results indicate that 1) in the presence of Cdc42, PAK2 autophosphorylation is a bipartite mechanism, with the regulatory domain autophosphorylated at multiple residues, whereas activation coincides with autophosphorylation of the catalytic domain at Thr-402; 2) the autophosphorylation reactions in regulatory domain are either a nonlimiting step or not required for activation of enzyme; 3) the autophosphorylation at site Thr-402 on the catalytic domain occurs by an intermolecular mechanism and is required for phosphorylation of exogenous substrates examined; 4) binding of the exogenous protein/peptide substrates at the active site of PAK2 has little or no effect on the autoactivation of PAK2, suggesting that multiple regions of PAK2 are involved in the enzyme-substrate recognition. The present method also provides a novel approach for studying autophosphorylation reactions. Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified, this new method would be particularly useful when the regulatory mechanisms of the reversible phosphorylation reaction toward certain enzymes are being assessed.

Protein kinases play a central role in cellular signal transduction, by transmitting biochemical information between activated membrane-bound receptors and physiological target proteins. In addition to phosphorylating other proteins, almost all protein kinases catalyse autophosphorylation reactions (i.e. reactions in which the kinase serves as its own substrate). The autophosphorylation reactions can be intramolecular or intermolecular. In the present study, a detailed kinetic analysis of the intermolecular autophosphorylation reaction is presented. On the basis of the kinetic equations, a new procedure is developed to evaluate the kinetic parameters of the autophosphorylation reaction. This method was used to analyse the intermolecular autophosphorylation of an S6/H4 kinase from human placenta. At a fixed ATP concentration of 0.125mM, the apparent catalytic-centre activity (turnover number; kcat) and apparent Michaelis—Menten constant (Km) for the autophosphorylation reaction were determined to be 0.91min-1 and 0.86μM respectively.

Cell-cell fusion generally requires cellular fusogenic proteins and actin-propelled membrane protrusions. However, the molecular connections between fusogens and the actin cytoskeleton remain unclear. Here, we show that the Caenorhabditis elegans fusogen EFF-1 and F-actin are enriched at the cortex of the post-embryonic fusing cells, and conditional mutations of WASP and Arp2/3 delay cell-cell fusion by impairing EFF-1 localization. Our affinity purification and mass spectrometry analyses determined that an actin-binding protein, spectraplakin/VAB-10A, binds to EFF-1. VAB-10A promotes cell-cell fusion by linking EFF-1 to the actin cytoskeleton. Conversely, EFF-1 enhanced the F-actin bundling activity of VAB-10A in vitro, and actin dynamics in the cortex were reduced in eff-1 or vab-10a mutants. Thus, cell-cell fusion is promoted by a positive feedback loop in which actin filaments that are crosslinked by spectraplakin to recruit fusogens to fusion sites are reinforced via fusogens, thereby increasing the probability of further fusogen accumulation to form fusion synapses.

Brassinosteroids (BRs) are essential steroid hormones that have crucial roles in plant growth and development. BRs are perceived by the cell-surface receptor-like kinase brassinosteroid insensitive 1 (BRI1). In the absence of BRs, the cytosolic kinase domain (KD) of BRI1 is inhibited by its auto-inhibitory carboxyl terminus, as well as by interacting with an inhibitor protein, BRI1 kinase inhibitor 1 (BKI1). How BR binding to the extracellular domain of BRI1 leads to activation of the KD and dissociation of BKI1 into the cytosol remains unclear. Here we report the crystal structure of BRI1 KD in complex with the interacting peptide derived from BKI1. We also provide biochemical evidence that BRI1-associated kinase 1 (BAK1) plays an essential role in initiating BR signaling. Steroid-dependent heterodimerization of BRI1 and BAK1 ectodomains brings their cytoplasmic KDs in the right orientation for competing with BKI1 and transphosphorylation.

Axin interactor, dorsalization-associated (Aida) was identified as a regulatory factor that utilizes its C-terminal region to interact with axis formation inhibitor (Axin). Aida abrogates the Axin-mediated Jun N-terminal kinase activation required for proper dorsalization during zebrafish embryonic development, and thus functions as a proventralization factor. Here, we report the structure of Aida C-terminal fragments, which adopt a conventional C2 domain topology. We also demonstrate that Aida can specifically bind to phosphoinositides in a Ca2+-independent manner, and is able to associate with the cell membrane via a novel positively charged surface, namely a basic loop. Mutation of the positively charged patch on the basic loop leads to destabilization of the Aida–membrane association or disruption of the Aida–Axin interaction, resulting in impaired Jun N-terminal kinase inhibition. Together, our findings provide a molecular basis for C2 domain-mediated Aida–membrane and Aida–Axin associations. Database The atomic coordinates and structure factors of the mouse Aida C2 domain (code: 2QZ5) and the zebrafish Aida C2 domain (code: 2QZQ) have been deposited in the Protein Data Bank (http://www.rcsb.org/) Structured digital abstract AIDA physically interacts with Axin by anti tag coimmunoprecipitation (View interaction)

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.

The kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the dephosphorylation of phosphorylase a by protein phosphatase-1 (ppase-1). On the basis of the kinetic equation of the substrate reaction in the presence of ppase-1, all the inactivation rate constants for the free enzyme and the enzyme-substrate(s) complexes have been determined. Binding of the allosteric substrate, glucose 1-phosphate, to one subunit of phosphorylase a protects completely against ppase-1 action on either the same subunit or the adjacent subunit, whereas binding of the non-allosteric substrate, glycogen, to one subunit protects this subunit partially, but has no effect on the modification on the neighbouring subunit. Analysis of the data suggests that the allosteric behaviour of phosphorylase a can be interpreted in terms of a modified concerted model. The present method also provides a novel approach for studying dephosphorylation reactions. Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified, this new method would be particularly useful when the regulatory mechanism of the reversible phosphorylation reaction toward certain enzymes is being assessed.

Substrate effects on the activation kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the presence of pCMB, all modification kinetic constants for the free enzyme and enzyme–substrate binary and ternary complexes have been determined. The results of the present study indicate that the modification of Chinese hamster dihydrofolate reductase by pCMB shows single-phase kinetics, and that changes in the enzyme activity and tertiary structure proceed simultaneously during the modification process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against modification by pCMB. In the presence of a saturating concentration of NADPH, the value of kcat for 7,8-dihydrofolate in the enzyme-catalysed reaction increased four-fold on modification of Cys-6, accompanied by a two-fold increase in Km for the modified enzyme. The utilization of the binding energy of a group to increase kcat rather than reduce Km implies that the full binding energy of the group is not realized in the formation of the enzyme–substrate complex, but is used to stabilize the enzyme–transition-state complex.

For the most frequently used two-site model, an exact binding equation is presented in terms of the total ligand concentration. This equation has been extended to analyze the spectroscopic titration experiment where the dilution of protein solution cannot be neglected, the displacement study, and the effect of non-specific binding. Thus, with a non-linear regression program, all unknown binding parameters can be determined correctly by fitting these equations to the experimental data without any data transformation. As an example of the use of the new equations, the experimental data for receptor-insulin binding were taken from literature and reanalyzed by using a non-linear regression data analysis program.

Both tyrosine kinase and topoisomerase II (TopII) are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK) detection kit using a horseradish peroxidase (HRP)-conjugated phosphotyrosine (pY20) antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 μmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 μM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P

A continuous spectrophotometric assay for the determination of protein phosphatase activity is presented. The assay incorporates the coupled enzyme system of Webb (M. R. Webb, 1992, Proc. Natl. Acad. Sci. USA 89, 4884-4887), which used purine nucleoside phosphorylase and the chromophoric substrate 7-methyl-6-thioguanosine for the quantitation of inorganic phosphate. The assay is exemplified and validated here for the phosphorylase phosphatase activity of protamine-stimulated protein phosphatase 2A1 (PP-2A1). The effects of reaction components on the activities of both PP-2A1 and purine nucleoside phosphorylase were studied. The application of the coupled assay system to kinetic analysis of the phosphorylase phosphatase activity of PP-2A1 and to the assay of the catalytic subunits of type 1 and 2A protein phosphatases and a recombinant type 1 catalytic subunit is demonstrated. The applicability of this coupled enzyme system to the assay of other protein phosphatases is discussed.

The energy sensor AMP-activated protein kinase (AMPK) is a heterotrimeric complex that is allosterically activated by AMP binding to the gamma subunit. Cocrystal structures of the mammalian AMPK core reveal occlusion of nucleotide-binding site 3 of the gamma subunit in the presence of ATP. However, site 3 is occupied in the presence of AMP. Mutagenesis studies indicate that sites 3 and 4 are important for AMPK allosteric activation.

The mitogen-activated protein kinase (MAPK) cascades play a pivotal role in a myriad of cellular functions. The specificity and efficiency of MAPK signaling are controlled by docking interactions between MAPKs and their cognate proteins. Many MAPK-interacting partners, including substrates, MAPK kinases, phosphatases, and scaffolding proteins, have linear sequence motifs that mediate the interaction with the common docking site on MAPKs. We report the crystal structure of p38α in complex with the MAPK binding domain (KBD) from MAPK phosphatase 5 (MKP5) at 2.7 Å resolution. In contrast to the well-known docking mode, the KBD binds p38α in a bipartite manner, in which two distinct helical regions of KBD engage the p38α docking site, which is situated on the back of the p38α active site. We also determined the crystal structure of the KBD of MKP7, which closely resembles the MKP5 KBD, suggesting that the mechanism of molecular recognition by the KBD of MKP5 is conserved in the cytoplasmic p38- and c-Jun N-terminal kinase-specific MKP subgroup. This previously unknown binding mode provides new insights into how MAPKs interact with their binding partners to achieve functional specificity.

We offer a novel graphical method for determining the number of essential sites in enzymes that contain multiple binding sites for a ligand. This method is applicable both to monomeric enzymes containing multiple "unspecific" sites (for protons, metal ions, etc.) and to oligomeric enzymes containing multiple "specific" sites (for substrates and their cognate analogues). The overall procedure is based on some of the intrinsic properties of the general rate equation for enzyme-catalyzed reactions involving multiple binding sites for Iigands as elaborated by W. G. Bardsley and R. E. Childs ( Biochem. J. , 149, 313-328, 1975). The experimental protocol involves measurement of initial rates of enzyme-catalyzed reactions either at varying concentrations of the substrate or at a fixed concentration of the substrate and varying concentrations of effectors (activator or inhibitor). The data are analyzed by plotting 1/ v [L] p versus [L] (for inhibition) and [L] p / v versus 1/[L] (for activation) for different (integral) increments of p (such as 0, 1, 2, 3, . . . etc.). According to the analytical procedure developed herein, the magnitude of p that yields a horizontal asymptote on these plots serves as the quantitative measure of the number of essential sites in enzyme molecules. By employing this procedure, we have been able to quantitatively ascertain the number of essential sites required for the activation or inhibition of a variety of monomeric and oligomeric enzymes. Among monomeric enzymes we have established that: (i) of the three binding sites for linoleic acid in the lipoxygenase molecule, one site is essential for catalysis, and the other two sites are inhibitory; (ii) of several plausible protonation sites in the α-chymotrypsin molecule, only one protonation site is required for the activation of the enzyme; and (iii) there are two inhibitory sites for guanidine-HCl per ribonuclease A molecule; the enzyme is fully inhibited upon binding of guanidine-HCl at any of these two sites. Among oligomericenzymes, we have discerned that: (i) the individual subunits of LDH and phosphorylase b are catalytically active, and (ii) the catalytic/functional unit of the creatine kinase molecule is likely to be the dimeric subunit. The theoretical details leading to the graphical analysis and its usage in delineating the functional stoichiometry of enzyme-ligand complexes are discussed.

Subclass III SnRK2s (SnRK2.6/2.3/2.2) are the key positive regulators of abscisic acid (ABA) signal transduction in Arabidopsis thaliana. The kinases, activated by ABA or osmotic stress, phosphorylate stress-related transcription factors and ion channels, which ultimately leads to the protection of plants from dehydration or high salinity. In the absence of stressors, SnRK2s are subject to negative regulation by group A protein phosphatase type 2Cs (PP2C), whereas the underlying molecular mechanism remains to be elucidated. Here we report the crystal structure of the kinase domain of SnRK2.6 at 2.6-Å resolution. Structure-guided biochemical analyses identified two distinct interfaces between SnRK2.6 and ABI1, a member of group A PP2Cs. Structural modeling suggested that the two interfaces lock SnRK2.6 and ABI1 in an orientation such that the activation loop of SnRK2.6 is posited to the catalytic site of ABI1 for dephosphorylation. These studies revealed the molecular basis for PP2Cs-mediated inhibition of SnRK2s and provided important insights into the downstream signal transduction of ABA.

A general procedure is described for determining the kinetic constants of the slow, tight-binding inhibition of enzyme-catalyzed reactions by analyzing the data of initial and steady-state rate. All unknown parameters can be determined from several simple, sequential calculations. This method is simple and rigorous. It is also applicable to the special case of slow-binding inhibition, where the total concentration of inhibitor is much higher than that of the enzyme.

The Wnt signaling plays pivotal roles in embryogenesis and cancer, and the three DIX domain-containing proteins, Dvl, Axin, and Ccd1, play distinct roles in the initiation and regulation of canonical Wnt signaling. Overexpressed Dvl has a tendency to form large polymers in a cytoplasmic punctate pattern, whereas the biologically active Dvl in fact forms low molecular weight oligomers. The molecular basis for how the polymeric sizes of Dvl proteins are controlled upon Wnt signaling remains unclear. Here we show that Ccd1 up-regulates canonical Wnt signaling via acting synergistically with Dvl. We determined the crystal structures of wild type Ccd1-DIX and mutant Dvl1-DIX(Y17D), which pack into "head-to-tail" helical filaments. Structural analyses reveal two sites crucial for intra-filament homo- and hetero-interaction and a third site for inter-filament homo-assembly. Systematic mutagenesis studies identified critical residues from all three sites required for Dvl homo-oligomerization, puncta formation, and stimulation of Wnt signaling. Remarkably, Ccd1 forms a hetero-complex with Dvl through the "head" of Dvl-DIX and the "tail" of Ccd1-DIX, depolymerizes Dvl homo-assembly, and thereby controls the size of Dvl polymer. These data together suggest a molecular mechanism for Ccd1-mediated Wnt activation in that Ccd1 converts latent polymeric Dvl to a biologically active oligomer(s).

SummaryThe plasma membrane-localized plant steroid hormone receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is quiescent in the absence of steroids, largely due to a negative regulator, BRI1 KINASE INHIBITOR 1 (BKI1). Here, we report that the steroid-induced, plasma membrane-dissociated and phosphorylated BKI1 also plays positive roles in BR signaling by interacting with a subset of 14-3-3 proteins. The cytosolic fraction of BKI1 carboxyl terminal region enhances BR signaling. Mutations of two serine residues in this region lead to reduced phosphorylation by the BRI1 kinase and constitutive plasma membrane localization. The 14-3-3 proteins can interact with the phosphorylated BKI1 through a motif that contains the two phosphorylation sites to release inhibition of BRI1 by BKI1. Meanwhile, the cytosolic BKI1 antagonizes the 14-3-3 s and enhances accumulation of BRI1 EMS SUPPRESSOR 1 (BES1)/BRASSINAZOLE RESISTANT 1 (BZR1) in the nucleus to regulate BR-responses.

The AMP-activated protein kinase (AMPK) is characterized by its ability to bind to AMP, which enables it to adjust enzymatic activity by sensing the cellular energy status and maintain the balance between ATP production and consumption in eukaryotic cells. It also has important roles in the regulation of cell growth and proliferation, and in the establishment and maintenance of cell polarity. These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes and cancer treatments. However, the regulatory mechanism of AMPK activity by AMP binding remains unsolved. Here we report the crystal structures of an unphosphorylated fragment of the AMPK alpha-subunit (KD-AID) from Schizosaccharomyces pombe that contains both the catalytic kinase domain and an autoinhibitory domain (AID), and of a phosphorylated kinase domain from Saccharomyces cerevisiae (Snf1-pKD). The AID binds, from the 'backside', to the hinge region of its kinase domain, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analyses indicate that AID binding might constrain the mobility of helix alphaC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation. Further in vitro kinetic studies demonstrate that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change in AMP concentration. The structural and biochemical data have shown the primary mechanism of AMPK autoinhibition and suggest a conformational switch model for AMPK activation by AMP.

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.

Negative cooperativity can be accounted for either by the pre-existent asymmetry model or by the ligand-induced sequential model. It is virtually impossible to deduce the mechanism of negatively cooperative interaction solely from the binding curves. Distinguishing between these two possible mechanisms for negative cooperativity usually requires experiments other than equilibrium binding. In the present communication, a kinetic method is proposed to distinguish between these two possible mechanisms for negative cooperativity. As an example of use of the new method, experimental data for the modification of creatine kinase by 5,5′-dithiobis-2-nitrobenzoic acid were taken from literature and reanalyzed by using the present method. The result indicates that under conditions in which creatine kinase forms the postulated ‘transition state analogue’ complex, the two subunits in the enzyme molecule have different tertiary structures and behave as different types of subunit.

Protein phosphorylation and limited proteolysis are two most common regulatory mechanisms involving the energy-dependent covalent modification of regulatory enzymes. In addition to modifying other proteins, many protein kinases and proteases catalyse automodification reactions (i.e. reactions in which the kinase or zymogen serves as its own substrate), and their activities are frequently regulated by other regulatory ligands. In the present study, a kinetic analysis of autocatalytic reaction modulated by regulatory ligands is presented. On the basis of the kinetic equation, a novel procedure is developed to evaluate the kinetic parameters of the reaction. As an example of an application of this method, the effects of calcium ions on the autoacatalytic activation of trypsinogen by trypsin is re-examined. The results indicate that the binding affinity for Ca2+-bound trypsinogen to trypsin is at least two orders of magnitude higher than that for Ca2+-free trypsinogen, and therefore that the effect of Ca2+ ions on Km* values for trypsinogen is very much greater than that for the model peptides. Based on the experimental results, one possible molecular mechanism has been proposed.

A novel spectrophotometric method to study the kinetics of the guanine nucleotide exchange factors-catalyzed reactions is presented. The method incorporates two coupling enzyme systems: (a). GTPase-activating protein which stimulates the intrinsic GTP hydrolysis reaction of small GTPases and (b). purine nucleotide phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for quantitation of the resultant inorganic phosphate. The continuous coupled enzyme system was used for characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors, Lbc and Dbl. Kinetic parameters obtained here show that there is no significant difference in kinetic mechanism of these GEFs in interaction with RhoA. The Michaelis-Menten constants were determined to be around 1micro M, and the rate constants k(cat) were around 0.1s(-1).

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cell. Mg 2+ ions play key roles in guanine nucleotide binding and in preserving the structural integrity of GTPases. We describe here the kinetics of the interaction of GTP with the Rho family small GTPase Cdc42 in the absence and presence of Mg 2+ . In contrast to the cases of Ras and Rab proteins, which require Mg 2+ for the nucleotide binding and intrinsic hydrolysis of GTP, our results show that in the absence of Mg 2+ , the binding affinity of GTP to Cdc42 is in the submicromolar concentration, and the Mg 2+ cofactor has only a minor effect on the Cdc42-catalyzed intrinsic hydrolysis rate of GTP. These results suggest that the intrinsic GTPase reaction mechanism of Cdc42 may differ significantly from that of other subfamily members of the Ras superfamily.

The inactive state of the small G protein Cdc42, the Cdc42.GDP.Mg(2+) ternary complex, was investigated using fluorescence, Mn(2+) substituted electron paramagnetic resonance, and (31)P nuclear magnetic resonance spectroscopy at various urea concentrations. The urea interaction with the protein was used to probe the binding state of GDP.Mg(2+) to Cdc42. Two binding states of the Cdc42.GDP.Mg(2+) ternary complex with different binding stability were observed. The two binding states were characterized by two sets of (31)P resonance of GDP phosphate groups, namely P(alpha) and P(beta), P('alpha), and P('beta). The high populated binding state I (P(alpha) and P(beta)) was more stable and less sensitive to the urea interaction. Yet the population of binding state II (P('alpha) and P('beta)) was lower, and the binding of GDP.Mg(2+) to Cdc42 in this state was more sensitive to the urea interaction. The release of GDP.Mg(2+) from the ternary complex in binding state II was faster than in state I.

The theoretical analysis has been presented for the kinetics of dissociating–associating enzyme-catalyzed reactions. On the basis of the kinetic equation of substrate reaction, a general procedure is developed for determining the kinetic constants of dissociating–associating enzyme reactions. By analyzing the experimental data of initial velocity and steady-state velocity as functions of enzyme and substrate concentration, all unknown kinetic parameters can be determined from several simple, sequential calculations. This method is simple and rigorous, and the required experiments may also not be difficult for most dissociating enzyme systems. Therefore, the present method should be a useful addition to the available methods for studying subunit dissociation of enzymes. In comparison to other physical methods, the advantage of this method is not only its usefulness in the study of self-associating reactions at very low protein concentration but its convenience in the study of substrate effects on subunit–subunit interactions.

The kinetic theory of substrate reaction during modification of enzyme activity has been applied to the study of inactivation kinetics of Chinese hamster dihydrofolate reductase by urea [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381–436]. On the basis of the kinetic equation of substrate reaction in the presence of urea, all microscopic kinetic constants for the free enzyme and enzyme–substrate binary and ternary complexes have been determined. The results of the present study indicate that the denaturation of dihydrofolate reductase by urea follows single-phase kinetics, and changes in enzyme activity and tertiary structure proceed simultaneously in the unfolding process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation, and enzyme–substrate complexes lose their activity less rapidly than the free enzyme.

The kinetic theory of substrate reaction during the modification of enzyme activity [Duggleby (1986) J. Theor. Biol. 123, 67–80; Wang and Tsou (1990) J. Theor. Biol. 142, 531–549] has been applied to a study of the inactivation kinetics of ribonuclease A by bromopyruvic acid. The results show that irreversible inhibition belongs to a non-competitive complexing type inhibition. On the basis of the kinetic equation of substrate reaction in the presence of the inhibitor, all microscopic kinetic constants for the free enzyme, the enzyme–substrate complex and the enzyme–product complex have been determined. The non-competitive inhibition type indicates that neither the substrate nor the product affects the binding of bromopyruvic acid to the enzyme and that the ionization state of His-119 may be the same in both the enzyme–substrate and the enzyme–product complexes.

Although the unfolding and refolding of proteins have been extensively studied in the literature, relatively few attempts have been made to see how many residues of the total residues of a certain amino acid in an enzyme can be modified without seriously affecting its folding. Based on a statistical analysis of the quantitative relationship between the extent of modification of protein functional groups and the decrease in their biological activity, a method proposed by Tsou (Sci. Sin. 1962, 11, 1535–1558) is widely used to determine the number of residues essential for the catalytic activity of modified proteins. In the present paper, Tsou's method is applied to determine the number of cysteine residues essential for the folding of creatine kinase. The thiol groups of the cysteine residues in fully unfolded creatine kinase were modified by 2-chloromercuri-4-nitrophenol (MNP). The relationship between the number of MNP-groups introduced and the recovery of activity after refolding was determined. Quantitative treatment of the data by Tsou's plot shows that among the cysteine residues modified in each subunit of creatine kinase, only three are essential for its folding.