Self-recognition and abstraction abilities in the common chimpanzee studied with distorting mirrors.

The reactions of chimpanzees to regular mirrors and the results of the standard Gallup mark test have been well documented. In addition to using the mark test to demonstrate self-recognition in a regular mirror, we exposed six female chimpanzees to mirrors that produced distorted or multiplied self-images. Their reactions to their self-images, in terms of mirror-guided self-referenced behaviors, indicated that correct assessment of the source of the mirror image was made by each subject in each of the mirrors. Recognition of a distorted self-image implies an ability for abstraction in the subjects in that the distortion must be rationalized before self-recognition occurs. The implications of these results in terms of illuminating the relative importance of feature and contingency of movement cues to self-recognition are discussed.

Rapid polyether cleavage via extracellular one-electron oxidation by a brown-rot basidiomycete

Fungi that cause brown rot of wood are essential biomass recyclers and also the principal agents of decay in wooden structures, but the extracellular mechanisms by which they degrade lignocellulose remain unknown. To test the hypothesis that brown-rot fungi use extracellular free radical oxidants as biodegradative tools, Gloeophyllum trabeum was examined for its ability to depolymerize an environmentally recalcitrant polyether, poly(ethylene oxide) (PEO), that cannot penetrate cell membranes. Analyses of degraded PEOs by gel permeation chromatography showed that the fungus cleaved PEO rapidly by an endo route. 13C NMR analyses of unlabeled and perdeuterated PEOs recovered from G. trabeum cultures showed that a major route for depolymerization was oxidative C—C bond cleavage, a reaction diagnostic for hydrogen abstraction from a PEO methylene group by a radical oxidant. Fenton reagent (Fe(II)/H2O2) oxidized PEO by the same route in vitro and therefore might account for PEO biodegradation if it is produced by the fungus, but the data do not rule out involvement of less reactive radicals. The reactivity and extrahyphal location of this PEO-degrading system suggest that its natural function is to participate in the brown rot of wood and that it may enable brown-rot fungi to degrade recalcitrant organopollutants.

Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

Muconate lactonizing enzyme (MLE), a component of the β-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the “enolase superfamily”) that catalyze the abstraction of the α-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-Å resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn2+-bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.

Structure and function of declarative and nondeclarative memory systems

This article reviews recent studies of memory systems in humans and nonhuman primates. Three major conclusions from recent work are that (i) the capacity for nondeclarative (nonconscious) learning can now be studied in a broad array of tasks that assess classification learning, perceptuomotor skill learning, artificial grammar learning, and prototype abstraction; (ii) cortical areas adjacent to the hippocampal formation, including entorhinal, perirhinal, and parahippocampal cortices, are an essential part of the medial temporal lobe memory system that supports declarative (conscious) memory; and (iii) in humans, bilateral damage limited to the hippocampal formation is nevertheless sufficient to produce severe anterograde amnesia and temporally graded retrograde amnesia covering as much as 25 years.

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.

Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains.

Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.

Designing safer chemicals: predicting the rates of metabolism of halogenated alkanes.

A computational model is presented that can be used as a tool in the design of safer chemicals. This model predicts the rate of hydrogen-atom abstraction by cytochrome P450 enzymes. Excellent correlations between biotransformation rates and the calculated activation energies (delta Hact) of the cytochrome P450-mediated hydrogen-atom abstractions were obtained for the in vitro biotransformation of six halogenated alkanes (1-fluoro-1,1,2,2-tetrachloroethane, 1,1-difluoro-1,2,2-trichloroethane, 1,1,1-trifluro-2,2-dichloroethane, 1,1,1,2-tetrafluoro-2-chloroethane, 1,1,1,2,2,-pentafluoroethane, and 2-bromo-2-chloro-1,1,1-trifluoroethane) with both rat and human enzyme preparations: In(rate, rat liver microsomes) = 44.99 - 1.79(delta Hact), r2 = 0.86; In(rate, human CYP2E1) = 46.99 - 1.77(delta Hact), r2 = 0.97 (rates are in nmol of product per min per nmol of cytochrome P450 and energies are in kcal/mol). Correlations were also obtained for five inhalation anesthetics (enflurane, sevoflurane, desflurane, methoxyflurane, and isoflurane) for both in vivo and in vitro metabolism by humans: In[F(-)]peak plasma = 42.87 - 1.57(delta Hact), r2 = 0.86. To our knowledge, these are the first in vivo human metabolic rates to be quantitatively predicted. Furthermore, this is one of the first examples where computational predictions and in vivo and in vitro data have been shown to agree in any species. The model presented herein provides an archetype for the methodology that may be used in the future design of safer chemicals, particularly hydrochlorofluorocarbons and inhalation anesthetics.