Lyle E. Carrington

Identification of mammalian-like purple acid phosphatases in a wide range of plants.

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.

Mechanism of allosteric regulation of transglutaminase 2 by GTP

Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the β-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.

Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle

BackgroundPurple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood.ResultsRed kidney bean purple acid phosphatase is a heterovalent enzyme with an Fe(III)Zn(II) center in the active site. Two new structures with bound sulfate (2.4 Å) and fluoride (2.2 Å) provide insight into the pre-catalytic phase of its reaction cycle and phosphorolysis. The sulfate-bound structure illustrates the significance of an extensive hydrogen bonding network in the second coordination sphere in initial substrate binding and orientation prior to hydrolysis. Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic μ-hydroxide present in the metal-bridging position. The fluoride-bound structure provides visual support for an activation mechanism for this μ-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity.ConclusionIn combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general.

THE INTERACTION OF CIMETIDINE WITH RAT LIVER MICROSOMES

The binding of cimetidine to rat liver microsomes in M/15 phosphate buffer, pH 7.9, has been investigated by difference spectroscopy and also by equilibrium partition studies, the latter method providing the more definitive characterization of the interaction in the pharmacologically relevant, low micromolar range of drug concn. In addition, the effect of cimetidine on the rate of dilution-induced displacement of [3H]cimetidine from rat liver microsomes has been used to justify consideration of the binding results in terms of two distinct and independent classes of microsomal site, governed by dissociation constants of 8.3 and 104 microM under the above conditions. By demonstrating unequivocally the existence of the stronger interaction, this investigation has provided an acceptable experimental basis for considering the undesired side effect of cimetidine in concomitant use with a number of other drugs to be the consequence of its inhibition of their monooxygenase-dependent metabolism.

Analysis of thermodynamic non-ideality in terms of protein solvation.

The effects of thermodynamic non-ideality on the forms of sedimentation equilibrium distributions for several isoelectric proteins have been analysed on the statistical-mechanical basis of excluded volume to obtain an estimate of the extent of protein solvation. Values of the effective solvation parameter delta are reported for ellipsoidal as well as spherical models of the proteins, taken to be rigid, impenetrable macromolecular structures. The dependence of the effective solvated radius upon protein molecular mass exhibits reasonable agreement with the relationship calculated for a model in which the unsolvated protein molecule is surrounded by a 0.52-nm solvation shell. Although the observation that this shell thickness corresponds to a double layer of water molecules may be of questionable relevance to mechanistic interpretation of protein hydration, it augurs well for the assignment of magnitudes to the second virial coefficients of putative complexes in the quantitative characterization of protein-protein interactions under conditions where effects of thermodynamic non-ideality cannot justifiably be neglected.

Crystallization and preliminary X-ray diffraction data for a purple acid phosphatase from sweet potato.

Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe-Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the first confirmed example of an Fe-Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 A and have the symmetry of space group P6(5)22, with one dimer per asymmetric unit. Diffraction data collected using a conventional X--ray source from a cryocooled crystal extend to 2.90 A resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre.

Ethanol and leucine oxidation—I. Leucine oxidation by the rat in vivo

The effect of ethanol upon the oxidation of leucine by the rat in vivo was determined. The rate of leucine oxidation was not significantly altered by chronic administration of ethanol (20% v/v solution as drinking water for 28 days). Ethanol administered acutely (8 g kg 0.73) significantly decreased leucine oxidation by the rat in vivo. This decrease appeared to be independent of a more general depression of oxidation metabolism. Decrease in leucine oxidation by ethanol is discussed in relation to the regulation of tissue leucine pool sizes in vivo.