Several hyperthermophilic organisms contain an unusual phosphatase that has dual activity toward inositol monophosphates and fructose 1,6-bisphosphate. The structure of the second member of this family, an FBPase/IMPase from Archaeoglobus fulgidus (AF2372), has been solved. This enzyme shares many kinetic and structural similarities with that of a previously solved enzyme from Methanococcus jannaschii (MJ0109). It also shows some kinetic differences in divalent metal ion binding as well as structural variations at the dimer interface that correlate with decreased thermal stability. The availability of different crystal forms allowed us to investigate the effect of the presence of ligands on the conformation of a mobile catalytic loop independently of the crystal packing. This conformational variability in AF2372 is compared with that observed in other members of this structural family that are sensitive or insensitive to submillimolar concentrations of Li ؉ . This analysis provides support for the previously proposed mechanism of catalysis involving three metal ions. A direct correlation of the loop conformation with strength of Li ؉ inhibition provides a useful system of classification for this extended family of enzymes.In mammalian cells, inositol monophosphatases are abundant cytosolic enzymes necessary to regenerate the supply of myo-inositol for the synthesis of phosphatidylinositols. However, in archaeal hyperthermophilic organisms, e.g. Archaeoglobus fulgidus, an IMPase 1 ortholog is thought to be used in the biosynthesis of a unique osmolyte, di-myo-inositol 1,1-phosphate (1). The archaeal enzymes have another twist as well.Besides catalyzing the hydrolysis of Ins-1-P (needed in response to thermal and salt stress) they also very specifically dephosphorylate FBP at C-1 (2). Enzymes with these characteristics exist as two distinct gene products in mammalian systems and several bacteria (3). The dual specificity of the enzyme from primitive hyperthermophiles strongly suggest that IMPase and FBPase enzymes may have evolved from the same gene product (2). Structural features of the IMPase/FBPase (MJ0109) from Methanococcus jannaschii (2) are similar to those of mammalian IMPase, inositol polyphosphase phosphatase (IPPase), FBPase, and the yeast enzyme Hal2. These enzymes form an extended family for which the general principles of chemical reactivity are the same (3). Two or three metal ions are necessary for catalysis to occur. From the structure of human IMPase with bound Ins-1-P and Gd 3ϩ , it was inferred that two metal ions were needed for catalysis (4). However, crystallographic data for pig kidney FBPase (5) and for MJ0109 (6) are consistent with a three-metal ion-assisted catalytic mechanism.There is strong biochemical evidence that Li ϩ inhibits IMPase in mammalian cells where a dose response to this ion correlates with a decrease in the intracellular pool of myoinositol (7). Li ϩ , by inhibiting mammalian IMPases, is thought to cause a depletion of inositol that attenuates the synthesis of phosphat...

X-ray structures of aspartate transcarbamoylase in the absence and presence of the first substrate carbamoyl phosphate are reported. These two structures in conjunction with in silico docking experiments provide snapshots of critical events in the function of the enzyme. The ordered substrate binding, observed experimentally, can now be structurally explained by a conformational change induced upon the binding of carbamoyl phosphate. This induced fit dramatically alters the electrostatics of the active site, creating a binding pocket for aspartate. Upon aspartate binding, a further change in electrostatics causes a second induced fit, the domain closure. This domain closure acts as a clamp that both facilitates catalysis by approximation and also initiates the global conformational change that manifests homotropic cooperativity.allosteric transition ͉ induced fit ͉ homotropic cooperativity E nzymes that exhibit positive cooperativity play a vital role in the regulation of the rates of key metabolic pathways by amplifying the response of a pathway to an effector molecule. In the case of aspartate transcarbamoylase (ATCase), substrateinduced domain closure triggers a quaternary conformational change that results in the observed homotropic cooperativity, one mechanism by which this enzyme controls the rate of de novo pyrimidine biosynthesis. Understanding the molecular features of domain closure not only provides insights into the mechanism of homotropic cooperativity but also demonstrates how ligandinduced domain closure can be used as part of catalytic mechanism of many enzymes (1, 2).The Escherichia coli ATCase is composed of six chains (M r 34,000 each) grouped into two trimeric catalytic subunits and six chains (M r 17,000 each) grouped into three dimeric regulatory subunits. The three active sites in the catalytic subunit are shared across the interface between adjacent chains (3, 4), whereas the regulatory subunits contain the binding sites for the heterotropic activator, ATP, as well as the heterotropic inhibitors, CTP and UTP. Each catalytic chain is composed of two structural domains, the carbamoyl phosphate (CP) domain (residues 1-135 and 292-310) and the L-aspartate (Asp) domain (residues 136-291), which contain the binding sites for CP and Asp, respectively. Each regulatory chain is also composed of two structural domains, the AL domain (residues 1-100) and the Zn domain (residues 101-153), which contain the binding sites for the allosteric effectors and the structural Zn, respectively. In mammals ATCase exists as a component of the multienzyme complex CAD (carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase) (5), and in humans, CAD has become a target for the development of antiproliferation drugs (6). The E. coli enzyme and the ATCase portion of CAD are 44% conserved, and all residues known to be involved in substrate binding and catalysis are conserved. Domain closure is triggered by the ordered binding of the substrates (7), with CP binding before Asp, and N-carbamoyl-L-...

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH > 7 in the presence of EGTA that is weakened as Ca 2؉ or Ba 2؉ are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba 2؉ was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA⅐Ca 2؉ surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca 2؉ modulation of PA⅐PLD binding involves protein aggregation that may be the critical interaction for activation.Mammalian phospholipase D enzymes have a complex intermediary role in many well characterized signal transduction pathways involving membrane-linked and cytosolic soluble signaling pathways (1). Most PLD 1 enzymes have a high affinity for anionic phospholipids, and all appear to require Ca 2ϩ for catalytic activity. Regulation of this class of enzymes is complex: protein kinase C (2), ARF (3), and Rho (2) proteins are activators of PLD. The lipophilic product of PLD cleavage, phosphatidic acid (PA), acts as a second messenger in cells and has been shown to activate phosphatidylinositol-specific phospholipase C-␥1 (4), inhibit adenylate cyclase (5), and mobilize intracellular Ca 2ϩ (6, 7). The PLD secreted by Streptomyces chromofuscus is considerably smaller than the eukaryotic enzymes, although like those enzymes it requires Ca 2ϩ for activity (8). The bacterial PLD is not involved in signal transduction but has a role in phosphate retrieval; it may also play a role in promoting infections of the organism. The S. chromofuscus PLD is an unusual phospholipase in that it does not exhibit "interfacial activation" (preference for micellar rather than monomeric short chain phospholipid substrate (9)) or "surface dilution" (dependence of enzyme specific activity on the mole fraction of substrate in a micelle or bilayer surface (10)) kinetics (11). A ping-pong-...

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Transgender youth face unique and complex issues as they confront cultural expectations of gender expression and how these fit with what is natural for them. Striving for balance, learning to cope, questioning, and eventually becoming comfortable with one's gender identity and sexual orientation are of paramount importance for healthy growth and development. Ineffective management of intense challenges over time without adequate social support places youth at risk for a number of unhealthy behaviors, including risk behaviors associated with acquiring HIV. This article explores early foundations of gender identity development, challenges in the development of transgender youth, and the limited data that exist on transgender youth and HIV risks. The concept of resilience is introduced as a counterbalancing area for assessment and intervention in practice and future research with transgender youth.