Enzyme catalysis: removing chemically 'essential' residues by site-directed mutagenesis.

Enzymatic catalysis relies on the action of the amino acid side chains arrayed in the enzyme active sites. Usually, only two or three 'essential' residues are directly involved in the bond making and breaking steps leading to product formation. For the past 20 years, enzymologists have been addressing the role of such residues by changing them into chemically inert side chains. Removal of an 'essential' group often does not abolish activity, but can significantly alter the catalytic mechanism. Such results underscore the sophistication of enzyme catalysis and the functional plasticity of enzyme active sites.

Dicephalic Parapagus Conjoined Twins in a Large Fruit-eating Bat, Genus Artibeus (Chiroptera, Phyllostomidae).

Conjoined twinning is an embryological anomaly rarely reported in wild mammals and with only two previous records in Chiroptera. Here, we report a case of dicephalic parapagus conjoined twins in the Neotropical phyllostomid genus Artibeus. These twins are males and present separated heads and necks, but a conjoined trunk with an expanded upper thoracic region. They developed two complete forelimbs and two complete hindlimbs, all laterally to the trunk. There is a volume in the upper midback and between the heads that resembles a third rudimentary medial forelimb, but X-ray images only suggest the presence of medial skeletal elements of the pectoral girdle (clavicle and scapulae) in this region. The X-ray images also show that vertebral columns run separated from head until the base of lumbar region, where they form a single structure. Using ultrasound images, we detected the presence of two similarly sized and apparently separated hearts. The accumulation of study cases like this will help in the understanding of patterns and process behind this phenomena, and collection material plays a key role in this context.

Time-resolved fluorescence of tryptophan synthase.

Time-resolved and steady-state fluorescence of the tryptophan synthase alpha 2 beta 2 complex and of the beta 2 dimer from Salmonella typhimurium were measured to characterize the conformational properties of the beta subunit in the presence and in the absence of the alpha subunit when the catalytic species internal aldimine, external aldimine and alpha-aminoacrylate Schiff bases were selectively accumulated within the beta active site. The fluorescence decay of the coenzyme pyridoxal 5'-phosphate, bound via a Schiff base in the beta subunit of the alpha 2 beta 2 complex (internal aldimine species), is accounted for by two lifetimes (2.9 and 0.9 ns) of almost equal fractional intensity that are slightly affected by pH. Accordingly, both the absorption and emission spectra were found to be pH independent. The emission properties of the internal aldimine in the beta 2 dimer are pH dependent, suggesting that the alpha-subunit binding alters the microenvironment of the beta-subunit active site. This conclusion is also supported by the emission of the single tryptophanyl residue of the enzyme (Trp-177 beta). In the reaction of L-serine with the alpha 2 beta 2 complex, the predominant catalytic intermediate is the external aldimine (lambda(max) = 422 nm) at pH 10, and the alpha-aminoacrylate (lambda(max) = 350 nm) at pH 7. The external aldimine exhibits a high fluorescence intensity at 500 nm that decays with a single lifetime of 6.2 ns in the alpha 2 beta 2 complex, at pH 10, and at a similar value in the beta 2 dimer. The emission properties of the external aldimine with respect to the internal aldimine, and the small effects induced by alpha-subunit binding indicate a shielding of the coenzyme and a stabilization of its excited state. In contrast, the short fluorescence lifetime (0.4 ns) and the weak fluorescence emission of the alpha-aminoacrylate Schiff base indicate an increase of non-radiative processes possibly due to a more tight coupling of this intermediate with the protein matrix with respect to the external aldimine. Whereas the internal aldimine is distributed in two tautomeric forms, both the external aldimine and the alpha-aminoacrylate are present in single conformational states with distinct structural and/or dynamic properties that may modulate regulatory intersubunit signals.

Ectoparasitic flies (Diptera, Streblidae) of bats (Chiroptera, Phyllostomidae) in an Atlantic Forest area, southeastern Brazil.

We studied infestation rates and parasite-host associations between streblid flies and phyllostomid bats in an Atlantic Forest area of Rio de Janeiro state, southeastern Brazil. We captured 301 individuals from seven Phyllostomidae bat species. Out of that total, 69 bats had been parasitised by nine Streblidae species; the most frequent species were Trichobius joblingi and Trichobius tiptoni. The species Paraeuctenodes longipes, associated with Anoura geoffroyi, was the most frequent species. The highest mean intensity was observed for Paraeuctenodes longipes, associated with A. geoffroyi, and Paratrichobius longicrus associated with Artibeus lituratus, both ectoparasite species with a mean intensity of five individuals per bat. Trichobius joblingi exhibited the highest mean abundance, which was over three on its host species. Streblid richness in the study area was similar to the richness found in other studies carried out in the Atlantic Forest. We observed that streblid richness in this biome depends more on inherent characteristics of each physiognomy and on the host-species than on the sampling effort.

Metapopulation in bats of Southern Brazil.

The purpose was to show that displacements, promoters of genetic diversity in metapopulations, increase the probability of survival of bat species adapted to medium and long-distance flights. Samples were taken in four forest fragments, distributed in three municipalities in northern Paraná, and the maximum distance between the studied areas is 20 km. A monthly sampling was performed for each fragment, for the period of July 2008 to June 2009. We used eight nets for collection which remained open during the first four hours of the night, totalling 192 hours during a year of study. The marking occurred from October 2008 to March 2009 and was accomplished through the use of anodised metal rings of four different colours. One hundred and fifty individuals were banded and since the first capture, four displacements were recorded. After five months of collecting and marking, one Carollia perspicillata was found three km away. Two Artibeus lituratus were recorded about 20 km from the marking place: the first one after 22 months and the second one after 24 months. Additionally, one Platyrrhinus lineatus was captured at about 20 km, after 26 months. As they moved around over considerable distances and are not monogamous, they mate with females of other fragments, exchanging genes and reducing or even avoiding inbreeding. Thus, populations of bats have the ability to increase genetic diversity in metapopulations, provided by displacements between the forest fragments. Species that behave like this are not vulnerable to isolation.

Sensitivity of populations of bats (Mammalia: Chiroptera) in relation to human development in northern Paraná, southern Brazil.

Most natural forests have been converted for human use, restricting biological life to small forest fragments. Many animals, including some species of bats are disappearing and the list of these species grows every day. It seems that the destruction of the habitat is one of its major causes. This study aimed to analyze how this community of bats was made up in environments with different sizes and quality of habitat. Data from studies conducted in the region of Londrina, Parana, Brazil, from 1982 to 2000 were used. Originally, this area was covered by a semi deciduous forest, especially Aspidosperma polyneuron (Apocynaceae), Ficus insipida (Moraceae), Euterpe edulis (Arecaceae), Croton floribundus (Euforbiaceae), and currently, only small remnants of the original vegetation still exist. The results showed a decline in the number of species caught in smaller areas compared to the largest remnant. In about 18 years of sampling, 42 species of bats were found in the region, representing 67% of the species that occur in Paraná and 24.4% in Brazil. There were two species of Noctilionidae; 21 of Phyllostoma; 11 Vespertilionidae and eight Molossidae. Eight of these were captured only in the largest fragment, Mata dos Godoy State Park (680 ha). Ten species had a low capture rate in the smaller areas with less than three individuals. Of the total sampled, 14 species were found in human buildings, and were able to tolerate modified environments, foraging and even using them as shelter. As the size of the forest area increases, there is a greater variety of ecological opportunities and their physical conditions become more stable, i.e., conditions favorable for growth and survival of a greater number of species. Forest fragmentation limits and creates subpopulations, preserving only long-lived K-strategist animals for some time, where the supporting capacity of the environment is a limiting factor. The reduction of habitats, species and genetic diversity resulting from human activities are endangering the future adaptability in natural ecosystems, which promotes the disappearance of low adaptive potential species.

Immunocytochemical study of gastrintestinal endocrine cells in insectivorous bats (Mammalia: Chiroptera).

The regional distribution and relative frequency of endocrine cells in the stomach and intestine of Phyllostomidae: Lonchorhina aurita and Molossidae: Molossus molossus bats were studied immunohistochemically. Three types of immunoreactive (IR) endocrine cells--to serotonin (5-HT), gastrin (GAS) and enteroglucagon (GLUC)--were found in the gastric mucosa and four types of IR cells were identified in the intestinal mucosa. This study showed an interespecfic difference in the regional distribution and relative frequency of endocrine cells in the Chiropteran alimentary tract.

Ectoparasitic flies (Diptera, Streblidae) of bats (Chiroptera, Phyllostomidae) in an Atlantic Forest area, southeastern Brazil / Moscas ectoparasitas (Diptera, Streblidae) de morcegos (Chiroptera, Phyllostomidae) numa área de Mata Atlântica, sudeste do Brasil

We studied infestation rates and parasite-host associations between streblid flies and phyllostomid bats in an Atlantic Forest area of Rio de Janeiro state, southeastern Brazil. We captured 301 individuals from seven Phyllostomidae bat species. Out of that total, 69 bats had been parasitised by nine Streblidae species; the most frequent species were Trichobius joblingi and Trichobius tiptoni. The species Paraeuctenodes longipes, associated with Anoura geoffroyi, was the most frequent species. The highest mean intensity was observed for Paraeuctenodes longipes, associated with A. geoffroyi, and Paratrichobius longicrus associated with Artibeus lituratus, both ectoparasite species with a mean intensity of five individuals per bat. Trichobius joblingi exhibited the highest mean abundance, which was over three on its host species. Streblid richness in the study area was similar to the richness found in other studies carried out in the Atlantic Forest. We observed that streblid richness in this biome depends more on inherent characteristics of each physiognomy and on the host-species than on the sampling effort.

Estudou-se as taxas de infestação e as associações parasita-hospedeiros de dípteros estreblídeos ectoparasitas de morcegos filostomídeos, em um fragmento de Mata Atlântica, no estado do Rio de Janeiro. Foram capturados 301 indivíduos de sete espécies de morcegos da família Phyllostomidae. Desse total, 69 morcegos encontravam-se parasitados com nove espécies de Streblidae, sendo Trichobius joblingi e Trichobius tiptoni as espécies mais freqüentes do total de estreblídeos coletados. Paraeuctenodes longipes, associada à Anoura geoffroyi foi a espécie mais prevalente. A maior intensidade média foi encontrada para Paraeuctenodes longipes, associada à A. geoffroyi e Paratrichobius longicrus associada à Artibeus lituratus, ambos com cinco ectoparasitas em média por morcego infestado. Trichobius joblingi apresentou a maior abundância média de infestação, que foi superior a três nas espécies de hospedeiros em que foi encontrada. A riqueza de estreblídeos da área de estudo é similar àquela obtida em outros estudos realizados na Mata Atlântica, e verificou-se que a riqueza de estreblídeos nesse bioma depende mais de outras características inerentes a cada fitofisionomia e à espécie hospedeira do que do esforço amostral de coleta.

Origins of the temperature dependence of hammerhead ribozyme catalysis.

The difficulties in interpreting the temperature dependence of protein enzyme reactions are well recognized. Here, the hammerhead ribozyme cleavage was investigated under single-turnover conditions between 0 and 60 degrees C as a model for RNA-catalyzed reactions. Under the adopted conditions, the chemical step appears to be rate-limiting. However, the observed rate of cleavage is affected by pre-catalytic equilibria involving deprotonation of an essential group and binding of at least one low-affinity Mg2+ion. Thus, the apparent entropy and enthalpy of activation include contributions from the temperature dependence of these equilibria, precluding a simple physical interpretation of the observed activation parameters. Similar pre-catalytic equilibria likely contribute to the observed activation parameters for ribozyme reactions in general. The Arrhenius plot for the hammerhead reaction is substantially curved over the temperature range considered, which suggests the occurrence of a conformational change of the ribozyme ground state around physiological temperatures.

Preferential activation of the 8-17 deoxyribozyme by Ca(2+) ions. Evidence for the identity of 8-17 with the catalytic domain of the Mg5 deoxyribozyme.

The 8-17 deoxyribozyme is a small RNA-cleaving DNA molecule of potential therapeutic interest. Here, the cleavage rates of 16 variants of the 8-17 deoxyribozyme were measured in the presence of different divalent metal ions. Despite the fact that 8-17 was originally selected in vitro for activity in the presence of Mg(2+) (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262-4266) nearly all the 8-17 variants exhibited substantially higher (up to 20-fold) reaction rates in Ca(2+) as compared with Mg(2+). This preference for calcium ions critically depended on the nucleoside residues at two specific positions of the deoxyribozyme core. The Ca(2+) specificity of 8-17 is strongly reminiscent of the properties of Mg5, an RNA phosphodiester-cleaving deoxyribozyme previously isolated by Faulhammer and Famulok (Faulhammer, D., and Famulok, M. (1996) Angew. Chem. Int. Ed. Engl. 35, 2837-2841). Indeed, analysis of the Mg5 sequence revealed the presence of a complete 8-17 motif, coincident with the conserved region of Mg5. An 8-17 deoxyribozyme modeled after the Mg5 conserved region displayed catalytic features comparable with those reported for the full-length Mg5 deoxyribozyme.

Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium.

Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan. We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site. The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band. Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme. The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges. The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer. The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay. In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational changes and subunit communication in tryptophan synthase: effect of substrates and substrate analogs.

The transmission of regulatory signals between the alpha- and beta-subunits of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium has been investigated by monitoring the luminescence properties of the enzyme in the presence and in the absence of the alpha-subunit ligand DL-alpha-glycerol 3-phosphate, the alpha- and beta-subunit substrate indole, and the beta-subunit substrate analog L-histidine. The beta-subunit contains as intrinsic probes Trp-177 and pyridoxal 5'-phosphate, whereas the alpha-subunit has been mutagenized by replacing Ala-129 with a Trp residue. In contrast to the inertness of L-histidine, DL-alpha-glycerol 3-phosphate was found (i) to alter the phosphorescence spectrum of Trp-129, (ii) to shift the fluorescence thermal quenching profile of both Trp-177 and coenzyme to higher temperature, (iii) to slow down the triplet decay kinetics of Trp-177 in fluid solution, and (iv) to affect the equilibrium between different conformations of the enzyme. These findings provide direct evidence that DL-alpha-glycerol 3-phosphate binding affects the structure of the alpha-subunit and, in the presence of coenzyme, induces a conformational change in the beta-subunit that leads to a considerably more rigid structure. As opposed to DL-alpha-glycerol 3-phosphate, the shortening of the phosphorescence lifetime upon indole binding suggests that this substrate increases structural fluctuations in the beta-subunit. Implications for the mechanism of the allosteric regulation between alpha- and beta-subunits are discussed.

Monovalent cations affect dynamic and functional properties of the tryptophan synthase alpha 2 beta 2 complex.

Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Their influence on the dynamic properties of the enzyme was probed by monitoring the phosphorescence decay of the unique Trp-177 beta, a residue located near the beta-active site, at the interface between alpha- and beta-subunits. In the presence of either Li+, Na+, Cs+, or NH4+, the phosphorescence decay is biphasic and the average lifetime increases indicating a decrease in the flexibility of the N-terminal domain of the beta-subunit. Since amplitudes but not lifetimes are affected, cations appear to shift the equilibrium between preexisting enzyme conformations. The effect on the reaction between indole and L-serine was studied by steady state kinetic methods at room temperature. We found that cations: (i) bind to the L-serine--enzyme derivatives with an apparent dissociation constant, measured as the concentration of cation corresponding to one-half of the maximal activity, that is in the millimolar range and decreases with ion size; (ii) increase kcat with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease KM for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the kcat/KM ratio by 20-40-fold. The influence on the equilibrium distribution between the external aldimine and the alpha-aminoacrylate, intermediates in the reaction of L-serine with the beta-subunits of the enzyme, was found to be cation-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases.

We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.

Microspectrophotometric studies on single crystals of the tryptophan synthase alpha 2 beta 2 complex demonstrate formation of enzyme-substrate intermediates.

Microspectrophotometry of single crystals of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium is used to compare the catalytic and regulatory properties of the enzyme in the soluble and crystalline states. Polarized absorption spectra demonstrate that chromophoric intermediates are formed between pyridoxal phosphate at the active site of the beta subunit and added substrates, substrate analogs, and reaction intermediate analogs. Although the crystalline and soluble forms of the enzyme produce some of the same enzyme-substrate intermediates, including Schiff base and quinonoid intermediates, in some cases the equilibrium distribution of these intermediates differs in the two states of the enzyme. Ligands which bind to the active site of the alpha subunit alter the distribution of intermediates formed at the active site of the beta subunit in both the crystalline and soluble states. The three-dimensional structures of the tryptophan synthase alpha 2 beta 2 complex and of a derivative with indole-3-propanol phosphate bound at the active site of the alpha subunit have recently been reported (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 264, 17857-17871). Our present findings help to establish experimental conditions for selecting defined intermediates for future x-ray crystallographic analysis of the alpha 2 beta 2 complex with ligands bound at the active sites of both alpha and beta subunits. These crystallographic studies should explain how catalysis occurs at the active site of the beta subunit and how the binding of a ligand to one active site affects the binding of a ligand to the other active site which is 25 A away.

Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation.

Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by the pro-Rp oxygen of phosphate 9 and by N7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The Rp-, but not Sp-, phosphorothioate reduces the cleavage rate by 10(3)-fold, and the rate can be fully restored by addition of low concentrations of Cd2+, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location approximately 20 A from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd2+ binds with a Kd of 25 microM in the ground state and a Kd of 2.5 nM in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.

Use of intrinsic binding energy for catalysis by an RNA enzyme.

The contribution of several individual ribozyme.substrate base pairs to binding and catalysis has been investigated using hammerhead ribozyme substrates that were truncated at their 3' or 5' ends. The base pairs at positions 1.1-2.1 and 15.2-16.2, which flank the conserved core, each contribute 10(4)-fold in the chemical step, without affecting substrate binding. In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step. These results suggest a "fraying model" in which each ribozyme.substrate helix can exist in either an unpaired ("open") state or a helical ("closed") state, with the closed state required for catalysis. The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form. Once the number of base pairs is sufficient to ensure that the closed helical state predominates, additional residues provide stabilization of the helix, and therefore increase binding, but have no further effect on the chemical step. Remarkably, the >5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energy expected for formation of a base pair. It is suggested that this unusually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is expressed in the transition state, where it is used for positioning. This extends the concept of "intrinsic binding energy" from protein to RNA enzymes, suggesting that intrinsic binding energy is a fundamental feature of biological catalysis.

Structure-function relationships in the hammerhead ribozyme probed by base rescue.

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

A core folding model for catalysis by the hammerhead ribozyme accounts for its extraordinary sensitivity to abasic mutations.

Introducing abasic nucleotides at each of 13 positions in the conserved core of the hammerhead ribozyme causes a large decrease in the extent of catalysis [Peracchi, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 11522]. This extreme sensitivity to structural defects is in contrast to the behavior of protein enzymes and larger ribozymes. Several additional differences in the behavior of the hammerhead relative to that of protein enzymes and larger ribozymes are described herein. The deleterious effects of the abasic mutations are not relieved by lowering the temperature, by increasing the concentration of monovalent or divalent metal ions, or by adding polyamines, in contrast to effects observed with protein enzymes and large RNA enzymes. In addition, the abasic mutations do not significantly weaken substrate binding. These results and previous observations are all accounted for by a "core folding" model in which the stable ground state structure of the hammerhead ribozyme complexed with the substrate is a partially folded state that must undergo an additional folding event to achieve its catalytic conformation. We propose that the peculiar behavior of the hammerhead arises because the limited structural interconnections in a small RNA enzyme do not allow the ground state to stably adopt the catalytic conformation; within the globally folded catalytic conformation, limited structural interconnections may further impair catalysis by hampering the precise alignment of active site functional groups. This behavior represents a basic manifestation of the well-recognized interconnection between folding and catalysis.

Chromosomal banding patterns in Akodon arviculoides (2n=14),Akodon sp.(2n=24 and 25), and two male hybrids with 19 chromosomes.

Chromosomal polymorphism resulting from two pericentric inversions in Akodon arviculoides (2n=14) has been described (YONENAGA, 1972a). In this paper the banding patterns are presented and identification of the inverted segments of the autosomal pairs 2 and 3 is made. The karyotype of Akodon sp., which varies in diploid number (2n-24 and 25), is described and shown to be due to the presence of a small submetacentric chromosome in the 2n=25 individuals. The karyotypes of two 2n=19 males studied show that they are hybrids between Akodon arviculoides (2n=14) and Akodon sp. (2n=24).