Mapping the transition state for ATP hydrolysis: implications for enzymatic catalysis

BACKGROUND: Phosphoryl transfer, typically involving high energy phosphate donors such as ATP, is the most common class of biological reactions. Despite this, the transition state for phosphoryl transfer from ATP in solution has not been systematically investigated. Characterization of the transition state for the uncatalyzed hydrolysis of ATP would provide a starting point for dissection of enzyme-catalyzed reactions. RESULTS: We examined phosphoryl transfer from ATP, GTP and pyrophosphate to a series of alcohols; these reactions are analogous to the phosphorylation of sugars and other biological alcohols and to the hydrolysis of ATP. The Br?nsted beta(nucleophile) value of 0.07 is small, indicating that there is little bond formation between the incoming nucleophile and the electrophilic phosphoryl group in the transition state. Coordination of Mg2+ has no measurable effect on this value. The Br?nsted beta(leaving group) value of -1.1 for phosphoryl transfer to water from a series of phosphoanhydrides is large and negative, suggesting that the bond between phosphorous and the leaving group oxygen is largely broken in the transition state. CONCLUSIONS: Uncatalyzed hydrolysis of ATP in solution occurs via a dissociative, metaphosphate-like transition state, with little bond formation between nucleophile and ATP and substantial cleavage of the bond between the gamma-phosphoryl moiety and the ADP leaving group. Bound Mg2+ does not perturb the dissociative nature of the transition state, contrary to proposals that enzyme-bound metal ions alter this structure. The simplest expectation for phosphoryl transfer at the active site of enzymes thus entails a dissociative transition state. These results provide a basis for analyzing catalytic mechanisms for phosphoryl transfer.     

The conserved asparagine and arginine are essential for catalysis of mammalian adenylyl cyclase

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C2 domain is catalytically active and the C1 domain is not; the role of the C1 domain is to promote the catalytic activity of the C2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C2 domains, but not among the C1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30-100-fold reduction in Kcat of adenylyl cyclase. However, the same mutations do not affect the Km for ATP, the half-maximal concentration (EC50) for the C2 domain of type II adenylyl cyclase to associate with the C1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC50 for forskolin to maximally activate enzyme activity with or without Gsalpha. This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in Kcat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.     

Inactivation of DNA-dependent protein kinase by protein kinase Cdelta: implications for apoptosis

Protein kinase Cdelta (PKCdelta) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCdelta in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCdelta (PKCdelta CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCdelta cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCdelta associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCdelta CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCdelta CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCdelta CF. These findings support the hypothesis that functional interactions between PKCdelta and DNA-PK contribute to DNA damage-induced apoptosis.     

Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme-MgADP-AlF3-diaminopelargonic acid and enzyme-MgADP-dethiobiotin-phosphate; implications for catalysis

The crystal structures of two complexes of dethiobiotin synthetase, enzyme-diaminopelargonic acid-MgADP-AlF3 and enzyme-dethiobiotin-MgADP-Pi, respectively, have been determined to 1.8 A resolution. In dethiobiotin synthetase, AlF3 together with carbamylated diaminopelargonic acid mimics the phosphorylated reaction intermediate rather than the transition state complex for phosphoryl transfer. Observed differences in the binding of substrate, diaminopelargonic acid, and the product, dethiobiotin, suggest considerable displacements of substrate atoms during the ring closure step of the catalytic reaction. In both complexes, two metal ions are observed at the active site, providing evidence for a two-metal mechanism for this enzyme.     

Transition state analogues for enzymes of nucleic acid metabolism

Knowledge of enzymatic transition states permits the logical design of transition state analogues. Kinetic isotope effects have been used to solve transition state structures for several N-ribosyltransferases. Nucleoside hydrolases from protozoan parasites show ribooxacarbenium ion character at their transition states, but with different extents of activation at the leaving group and the oxacarbenium ion. Transition state analogues are designed to capture these interactions and provide isozyme-specific inhibitors. Ricin A-chain is an RNA N-ribohydrolase for a single site on 28S rRNA. Its transition state resembles a fully dissociated ribooxacarbenium ion. A transition state analogue for ricin A-chain mimics the fully dissociated purine and cationic ribosyl transition state. The transition state for human purine nucleoside phosphorylase (PNP) is more dissociated than for the bovine enzyme. Immucillin-H, a powerful transition state inhibitor for human PNP, has entered clinical trials as an anti T-cell agent.     

The structure of human interferon-beta: implications for activity

Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-beta (HuIFN-beta), in particular in relation to its activity. The recently determined crystal structure of HuIFN-beta provides a framework for understanding of the mechanism of differentiation of type I IFNs by their common receptor. Insights are generated by comparison with the structures of other type I IFNs and from the interpretation of existing mutagenesis data. The details of the observed carbohydrate structure, together with biochemical data, implicate the glycosylation of HuIFN-beta, which is uncommon among type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein. Finally, these structural implications are discussed in the context of the clinical use of HuIFN-beta.     

Regulation of type-II calmodulin kinase: functional implications

Calmodulin-kinase II (CaM kinase) is a calcium/calmodulin-dependent protein kinase which is highly enriched in the nervous system and mediates many of calcium's actions. Regulation of CaM kinase activity plays an important role in modulating synaptic transmission, synaptic plasticity and in neuropathology. Primary regulation of CaM kinase occurs via changes in intracellular calcium concentrations. Increased calcium stimulates protein kinase activity and induces autophosphorylation. Autophosphorylation of CaM kinase at specific sites results in altered activity and responsiveness to subsequent changes in calcium concentrations. Intracellular translocation of CaM kinase also appears to result from autophosphorylation. These mechanisms of regulation play an important role in synaptic plasticity (e.g., Aplysia ganglia), status epilepticus and cerebral ischemia. Long-lasting alterations in the expression of CaM kinase have been demonstrated in the kindling model of epilepsy and in monocular deprivation and therefore modulation of gene expression, in addition to autophosphorylation and translocation, appears to be another important mechanism of regulating CaM kinase activity.     

Protein farnesyltransferase: structure and implications for substrate binding

The rat protein farnesyltransferase crystal structure has been solved by multiple isomorphous replacement methods at a resolution of 2.75 A. The three-dimensional structure, together with recent data on the effects of several mutations, led us to propose a model for substrate binding which differs from the model presented by Park et al. based on their independent structure determination [Park, H. -W., Boduluri, S. R., Moomaw, J. F., Casey, P. J., and Beese, L. S. (1997) Science 275, 1800-1804]. Both farnesyl diphosphate and peptide substrates can be accommodated in the hydrophobic active-site barrel, with the sole charged residue inside the barrel, Arg202 of the beta-subunit, forming a salt bridge with the negatively charged carboxy terminus of peptide substrates. Our proposals are based in part on the observation of electron density in the active site which can be modeled as bound farnesyl diphosphate carried through the enzyme purification. In addition, our model explains in structural terms the results of mutational studies which have identified several residues critical for substrate specificity and catalysis.     

NMR solution structure of the lead-dependent ribozyme: evidence for dynamics in RNA catalysis

The function of social calls emitted by foraging bats has received little study. Here we use observations of free-ranging greater spear-nosed bats, Phyllostomus hastatus, and field playbacks to determine whether audible, broad-band 'screech' calls attract mates, warn conspecifics or influence access to food. Five lines of evidence suggest that screech calls enable adult females from the same roosting group to fly together from the day roost to feeding sites. (1) Seasonal differences in diet influenced the rate of screech calling recorded outside the cave roost, as well as how often bats departed together. Bats called more often and flew in larger groups when feeding on a concentrated resource, balsa, Ochroma lagopus, flowers, in winter than on more dispersed Cecropia peltata fruit in spring. (2) Observations of bats flying outside the cave, in flyways and at feeding sites indicated that screech calls occurred more often when bats flew in groups than alone. (3) Females from the same roosting group were netted at the same feeding site, sometimes simultaneously, several kilometres from the cave. (4) Calling colour-marked adult females outside the cave were joined by a female group member, both on initial departures and on second foraging trips, more often than non-calling bats. (5) Playbacks attracted conspecifics at roost and feeding sites. Screech calls appear to function as contact calls that recruit and coordinate foraging among group members. We postulate that females benefit from foraging with unrelated roost-mates because they can defend feeding sites more effectively. Copyright 1998 The Association for the Study of Animal Behaviour.     

A theoretical study of torsional flexibility in the active site of aspartic proteinases: implications for catalysis

We have performed ab initio Hartree-Fock self-consistent field calculations on the active site of endothiapepsin. The active site was modeled as a formic acid/formate anion moiety (representing the catalytic aspartates, Asp-32 and -215) and a bound water molecule. Residues Gly-34, Ser-35, Gly-217, and Thr-218, which all form hydrogen bonds to the active site, were modeled using formamide and methanol molecules. The water molecule, which is generally believed to function as the attacking nucleophile in catalysis, was allowed to bind to the active site in four distinct configurations. The geometry of each configuration was optimized using two basis sets (4-31G and 4-31G*). The results indicate that in the native enzyme the nucleophilic water is bound in a catalytically inert configuration. However, by rotating the carboxyl group of Asp-32 by about 90 degrees the water molecule can be reorientated to attack the scissile bond of the substrate. A model of the bound enzyme-substrate complex was constructed from the crystal structure of a difluorostatone inhibitor complexed with endothiapepsin. This model suggests that the substrate itself initiates the reorientation of the nucleophilic water immediately prior to catalysis by forcing the carboxyl group of Asp-32 to rotate. The theoretical results predict that the active site of endothiapepsin undergoes a large distortion during substrate binding and this observation has been used to explain some of the kinetics results which have been reported for mutant aspartic proteinases.     

Mutation of the distal arginine in Coprinus cinereus peroxidase--structural implications

Heme peroxidases of prokaryotic, plant and fungal origin share the essential His and Arg catalytic residues of the distal cavity and a proximal His bound to heme iron. Spectroscopic techniques, in contrast to X-ray crystallography, are well suited to detect the precise structure, spin and coordination states of the heme as influenced by its near environment. Resonance Raman and electronic absorption spectra obtained at various pH values for Fe3+ and Fe2+ forms of distal Arg51 mutants of the fungal Coprinus cinereus peroxidase are reported, together with the fluoride adducts at pH 5.0. This basic catalytic residue has been replaced by the aliphatic residue Leu, the polar residues Asn and Gln and the basic residue Lys (Arg51-->Leu, Asn, Gln, and Lys, respectively). These mutations cause changes in the coordination and spin states of the heme iron, and in the v(Fe-Im) stretching frequency. The variations are explained in terms of pH-dependent changes, charge location, size and hydrogen-bonding acceptor/donor properties of the residue at position 51. The present work indicates that the hydrogen-bond capability of the residue in position 51 influences the occupancy of water molecules in the distal cavity and the ability to form stable complexes between anionic ligands and the heme Fe atom.     

Modeling of noncatalytic fluid-solid reactions: The quasi-steady state assumption

An isothermal, topochemical model for noncatalytic fluid-solid reactions, in spherical pellets, is formulated, solved numerically, and investigated experimentally. The equations are transformed into dimensionless form in order to introduce characteristic dimensionless groups. The dimensionless groups represent the relative effects of the diffusional, fluid film transport, and chemical reaction rates on the system, as well as the ratio of the molar density of the reactant fluid to that of the reactant solid. The model is solved with and without the quasi-steady state assumption in order to investigate the effects of the values of the dimensionless groups on the error introduced by this assumption. The model, without the quasi-steady state assumption, is shown to be an accurate representation of a resin exchange system, and reduces to the quasi-steady state model for appropriate values of the dimensionless groups.     

Crystal structure of CTP-ligated T state aspartate transcarbamoylase at 2.5 A resolution: implications for ATCase mutants and the mechanism of negative cooperativity

The X-ray crystal structure of CTP-ligated T state aspartate transcarbamoylase has been refined to an R factor of 0.182 at 2.5 A resolution using the computer program X-PLOR. The structure contains 81 sites for solvent and has rms deviations from ideality in bond lengths and bond angles of 0.018 A and 3.722 degrees, respectively. The cytosine base of CTP interacts with the main chain carbonyl oxygens of rTyr-89 and rIle-12, the main chain NH of rIle-12, and the amino group of rLys-60. The ribose hydroxyls form polar contacts with the amino group of rLys-60, a carboxylate oxygen of rAsp-19, and the main chain carbonyl oxygen of rVal-9. The phosphate oxygens of CTP interact with the amino group of rLys-94, the hydroxyl of rThr-82, and an imidazole nitrogen of rHis-20. Recent mutagenesis experiments evaluated in parallel with the structure reported here indicate that alterations in the hydrogen bonding environment of the side chain of rAsn-111 may be responsible for the homotropic behavior of the pAR5 mutant of ATCase. The location of the first seven residues of the regulatory chain has been identified for the first time in a refined ATCase crystal structure, and the proximity of this portion of the regulatory chain to the allosteric site suggests a potential role for these residues in nucleotide binding to the enzyme. Finally, a series of amino acid side chain rearrangements leading from the R1 CTP allosteric to the R6 CTP allosteric site has been identified which may constitute the molecular mechanism of distinct CTP binding sites on ATCase.     

Cuban value structure: Treatment implications.

Discusses the relationship between cultural variables and psychosocial treatment. It is assumed that in order for psychosocial treatment to be acceptable and effective with a client population, it must be sensitive to the cultural characteristics of that population. The paradigm of planning therapy according to the cultural characteristics of a population is illustrated for Cuban immigrant adolescents. To investigate cultural variables, a Value Orientations Scale was developed based on the work of F. R. Kluckhohn and F. L. Strodtbeck (1961) using 220 Cuban immigrant, 65 White American, 28 Black American, and 12 non-Cuban Latin 15–77 yr old Ss. Four factorially derived subscales were obtained. When 56 Cuban immigrant and 152 Anglo-American adolescents were compared along the Value Orientations Scale, the Cubans tended to prefer lineality, subjugation to nature, present time, and not to endorse idealized humanistic values, whereas the Americans tended to prefer individuality, mastery over nature, future time, and to endorse idealized humanistic values. The implications of these value differences for the delivery of mental health treatment are discussed. (22 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Detection of global structure in Glass patterns: implications for form vision

Glass (Nature 1969;223:578-580) patterns are random dot stimuli that generate a percept of global structure. To study the mechanisms underlying this global form perception, concentric, radial, hyperbolic, and parallel Glass patterns were constructed. Thresholds for detecting each type of pattern were measured by degrading the patterns through the addition of noise. Concentric patterns yielded the lowest thresholds for all subjects, while radial and hyperbolic patterns produced somewhat higher thresholds. For all subjects the parallel patterns produced the highest thresholds. Threshold measurements as a function of the area containing pattern structure provided evidence for global pooling of orientation information in the detection of radial and concentric Glass patterns but only local pooling in the detection of parallel patterns. Monte-Carlo simulations demonstrate that plausible neural models can accurately predict the data. These models indicate that the visual system contains networks that pool orientation information within regions 3.5-4.5 degrees in diameter in central vision. This pooling is organized to extract cross-shaped, X-shaped, and quasi-circular forms from the retinal image. The results are in good agreement with recent single unit physiology of primate area V4, an intermediate level of the form vision pathway.     

Tyrosine kinase activity modulates catalysis and translocation of cellular 5-lipoxygenase

Tyrosine kinase activity, a determinant of Src homology domain interactions, has a prominent effect on cellular localization and catalysis by 5-lipoxygenase. Six separate inhibitors of tyrosine kinase each inhibited 5(S)-hydroxyeicosatetraenoic acid formation by HL-60 cells stimulated with calcium ionophore, in the presence or absence of exogenous arachidonic acid substrate, indicating that they modulated cellular 5-lipoxygenase activity. The tyrosine kinase inhibitors also blocked the translocation of 5-lipoxygenase from cytosol to membranes during cellular activation, consistent with their effects on its catalytic activity. These results fit a model which postulates that Src homology domain interactions are a molecular determinant of the processes which coordinate the subcellular localization and functions of 5-lipoxygenase. In addition, we demonstrate that activated leukocytes contain two molecularly distinct forms of 5-lipoxygenase: a phosphorylated form and a nonphosphorylated form. In activated HL-60 cells the pool of phosphorylated 5-lipoxygenase accumulates in the nuclear fraction, not with the membrane or cytosolic fractions. The amount of phosphorylated 5-lipoxygenase is a small fraction of the total. Overall, equilibrium reactions involving the nuclear localizing sequence, the proline-rich SH3 binding motif, and the phosphorylation state of 5-lipoxygenase may each influence its partnership with other cellular proteins and any novel functions derived from such partnerships.     

Tyrosine kinase: implications in tumor pathology and therapeutic perspectives

PURPOSE: Both generalist and pulmonologist physicians care for patients with severe chronic obstructive pulmonary disease (COPD). We studied patients hospitalized with severe COPD to explore whether supervision of care by pulmonologists is associated with greater costs or better survival. SUBJECTS AND METHODS: We studied 866 adults with severe COPD enrolled in the Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatments (SUPPORT), a prospective study at five academic medical centers. Patients were admitted to the hospital or transferred to an intensive care setting for treatment of severe COPD, defined by hypoxia (PaO2 <60 mm Hg) and hypercapnia (PaCO2 >50 mm Hg) or hypercapnia alone if on supplemental oxygen. Resource intensity was measured using a modified version of the Therapeutic Intervention Scoring System and estimated hospital costs. To account for differences in the patient case mix, propensity scores were developed to represent each patient's probability of having a pulmonologist as attending physician and each patient's probability of being in an intensive care unit (ICU) at study admission. RESULTS: Of the 866 patients studied, 512 had generalists and 354 pulmonologists as their attending physicians. The median patient age was 70 years; 52% were male; 14% died within 30 days. After adjusting for baseline differences in patient characteristics, there were no differences in resource intensity and hospital costs in those treated by pulmonologists or generalists. Adjusted average resource intensity scores for the entire hospitalization were 16.5 for pulmonologists and 17.0 for generalists (P = 0.34). Estimated hospital costs were the same ($6,400) for patients treated by pulmonologists and generalists (P = 0.99). Patients with pulmonologists as attending physicians did not experience better survival. Comparing patients of pulmonologists to patients of generalists, the adjusted hazard ratio for 30-day mortality was 1.6 (95% confidence interval: 0.98, 2.5); the hazard ratio for 180-day mortality was 1.2 (0.9, 1.7). CONCLUSIONS: Our findings suggest that for patients hospitalized with exacerbation of severe COPD, those with pulmonologist attending physicians do not have higher hospital resource use or better survival than those with generalist attending physicians.     

Thymidylate synthase: structure, inhibition, and strained conformations during catalysis

Thymidylate synthase (TS) is a long-standing target for chemotherapeutic agents because of its central role in DNA synthesis, and it is also of interest because of its rich mechanistic features. The reaction catalyzed by TS is the methylation of dUMP, with the transferred methyl group provided by the cofactor methylenetetrahydrofolate (CH2THF). Recently, several crystal structure determinations and mechanistic studies have led to a deeper understanding of the TS reaction mechanism, and address the role of conformational change in TS catalysis and inhibition. Included among these structures are complexes of TS bound to substrate dUMP; cofactor CH2THF; the nucleotide analogs 5-fluoro-dUMP, 5-nitro-dUMP and dGMP; and the promising antifolates BW1843, ZD1694, and AG337. From these studies, a picture of TS emerges where ligand-induced conformational changes play key roles in catalysis by straining the thiol adduct that occurs during the reaction; by protecting the highly reactive reaction intermediates; and by providing a means to stabilize a high-energy conformer of the cofactor after initial binding of a low-energy conformer. The best inhibitors of TS also induce and stabilize a conformational change in TS. One inhibitor, BW1843, distorts the active site on binding, and intercalates into a hydrophobic patch between two mobile subdomains in the protein. Also discussed are recent developments in the cell biology and regulation of eukaryotic TS and the use of structure-based drug design in the development of the antifolates currently in clinical trial for the treatment of cancer.     

Welfare reform and women's health: review of the literature and implications for state policy

In August 1996, the Personal Responsibility Work Opportunity Reconciliation Act (P.L. 104-193) was signed into law, ending a 60-year federal entitlement guaranteeing families some basic level of assistance during periods of economic hardship. Several components of this new legislation have the potential to impact upon the health and well-being of women and children. We summarize studies examining the relationship between welfare participation and physical and mental well-being of women and what is known about the effects of poverty on health; the patterns of employment among welfare participants and the health consequences of low-wage work on women; domestic violence among welfare recipients; the potential health consequences of the provisions of the new Temporary Assistance to Needy Families (TANF) program for women's and adolescent health; and the consequences of the new TANF provisions for the health and well-being of immigrant women. We discuss the implications for policy makers in monitoring and minimizing the negative impact of welfare reforms on women's health and well-being.