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The limits of enzyme specificity and the evolution of metabolism
Full text linkThe substrate specificity of enzymes is bound to be imperfect, because of unavoidable physicochemical limits. In extant metabolic enzymes, furthermore, such limits are seldom approached, suggesting that the degree of specificity of these enzymes, on average, is much lower than could be attained. During biological evolution, the activity of a single enzyme with available alternative substrates may be preserved to a significant or even substantial level for different reasons – for example when the alternative reaction contributes to fitness, or when its undesirable products are nevertheless dispatched by metabolite repair enzymes. In turn, the widespread occurrence of promiscuous reactions is a consistent source of metabolic ‘messiness’, from which both liabilities and opportunities ensue in the evolution of metabolic systems
DNA catalysis: potential, limitations, open questions
No full textIn vitro selection methods are shedding light on the surprising functional versatility of DNA, and in particular on its catalytic aptitudes. The study of DNA enzymes (deoxyribozymes) helps address the principles and the limits of nucleic acid catalysis, while also providing useful tools for biotechnology and nanotechnology
Prospects for antiviral ribozymes and deoxyribozymes
No full textRibozymes and deoxyribozymes (collectively referred to as nucleic acid enzymes) can be designed to cleave substrate mRNAs in a sequence-specific manner, and thus represent a potentially important tool to inhibit selectively the expression of deleterious genes. Nucleic acid enzymes can be delivered to cells either as genes encoding RNA enzymes (ribozymes), or exogenously as in vitro produced agents. This review focuses in particular on the 'exogenous application' of ribozymes and deoxyribozymes as antiviral drugs.
In the past few years, the therapeutic development of ribozymes and deoxyribozymes has encountered a variety of problems, several of which have now been solved thanks to a massive amount of research. Much progress has been made towards understanding the structure and mechanism of these catalysts, improving their stability and effectiveness in vivo, and investigating their clinical usage. Despite this, it is not yet clear whether these molecules can be developed into clinically useful pharmaceutical preparations. To address the long-term prospects of this class of therapeutics, it is necessary to consider their intrinsic capabilities and limits, the developmental difficulties that they still face and the comparative advantages and disadvantages they may offer with respect to other oligonucleotide-based therapeutic approaches
Preferential activation of the 8-17 deoxyribozyme by Ca2+ ions. Evidence for the identity of 8-17 with the catalytic domain of the Mg5 deoxyribozyme
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How (and why) to revive a dead enzyme: the power of chemical rescue
No full textChemical rescue is an experimental strategy whereby the activity of a mutant enzyme is restored upon the addition of small exogenous compounds, which somehow surrogate the function of the mutated residue. These molecules become in effect "probes" of the chemical and structural requirements for efficient catalysis by the mutant enzyme. Entire batteries of small compounds can be tested for rescue, making it easier to implement the methods of physical organic chemistry (such as Brønsted analysis) to the study of enzymatic catalysis. To date, chemical rescue has been employed to address enzyme mechanism in over a hundred studies, helping to identify catalytic residues, to better outline their roles and to probe the structural and functional context in which catalysis occurs. Recently, some researchers have explored the use of this strategy to modulate the activity of specific enzymes in vivo, as a tool for dissecting complex cellular processes. These studies have also raised the possibility that chemical rescue might one day be applied in theraphy, for the reactivation of genetically defective enzymes. The present review illustrates the power, the pitfalls and the perspectives of this approach
Dissecting the hybridization of oligonucleotides to structured complementary sequences
No full textBackground: When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. Methods: I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and > 20 complementary oligonucleotides ('antisenses'). Results: Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. Conclusions: The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. General significance: While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process
The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families
Full text linkAbstract Background - Enzymes that depend on vitamin B6 (and in particular on its metabolically active form, pyridoxal 5'-phosphate, PLP) are of great relevance to biology and medicine, as they catalyze a wide variety of biochemical reactions mainly involving amino acid substrates. Although PLP-dependent enzymes belong to a small number of independent evolutionary lineages, they encompass more than 160 distinct catalytic functions, thus representing a striking example of divergent evolution. The importance and remarkable versatility of these enzymes, as well as the difficulties in their functional classification, create a need for an integrated source of information about them. Description - The B6 database http://bioinformatics.unipr.it/B6db contains documented B6-dependent activities and the relevant protein families, defined as monophyletic groups of sequences possessing the same enzymatic function. One or more families were associated to each of 121 PLP-dependent activities with known sequences. Hidden Markov models (HMMs) were built from family alignments and incorporated in the database. These HMMs can be used for the functional classification of PLP-dependent enzymes in genomic sets of predicted protein sequences. An example of such analyses (a census of human genes coding for PLP-dependent enzymes) is provided here, whereas many more are accessible through the database itself. Conclusion - The B6 database is a curated repository of biochemical and molecular information about an important group of enzymes. This information is logically organized and available for computational analyses, providing a key resource for the identification, classification and comparative analysis of B6-dependent enzymes.</p
A subfamily of PLP-dependent enzymes specialized in handling terminal amines
Full text linkThe present review focuses on a subfamily of pyridoxal phosphate (PLP)-dependent enzymes, belonging to the broader fold-type I structural group and whose archetypes can be considered ornithine δ-transaminase and γ-aminobutyrate transaminase. These proteins were originally christened “subgroup-II aminotransferases” (AT-II) but are very often referred to as “class-III aminotransferases”. As names suggest, the subgroup includes mainly transaminases, with just a few interesting exceptions. However, at variance with most other PLP-dependent enzymes, catalysts in this subfamily seem specialized at utilizing substrates whose amino function is not adjacent to a carboxylate group.
AT-II enzymes are widespread in biology and play mostly catabolic roles. Furthermore, today several transaminases in this group are being used as bioorganic tools for the asymmetric synthesis of chiral amines. We present an overview of the biochemical and structural features of these enzymes, illustrating how they are distinctive and how they compare with those of the other fold-type I enzymes
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