An amino acid as a cofactor for a catalytic polynucleotide

Abstract

Natural ribozymes require metal ion cofactors that aid both in structural folding and in chemical catalysis. In contrast, many protein enzymes produce dramatic rate enhancements using only the chemical groups that are supplied by their constituent amino acids. This fact is widely viewed as the most important feature that makes protein a superior polymer for the construction of biological catalysts. Herein we report the in vitro selection of a catalytic DNA that uses histidine as an active component for an RNA cleavage reaction. An optimized deoxyribozyme from this selection requires L-histidine or a closely related analog to catalyze RNA phosphoester cleavage, producing a rate enhancement of Ϸ1-million-fold over the rate of substrate cleavage in the absence of enzyme. Kinetic analysis indicates that a DNA-histidine complex may perform a reaction that is analogous to the first step of the proposed catalytic mechanism of RNase A, in which the imidazole group of histidine serves as a general base catalyst. Similarly, ribozymes of the ''RNA world'' may have used amino acids and other small organic cofactors to expand their otherwise limited catalytic potential.The ''RNA world'' theory for the origins of life holds that RNA was able to replicate in the absence of DNA and proteins (1). Some manifestations of this theory (1-3) include the view that, during the latter stages of the RNA world, ribozymes were not primitive and ineffectual but were directing a complex metabolic state replete with RNA and DNA synthesis machinery and a host of RNA enzymes necessary to catalyze supportive chemical reactions. Proponents of this view argue that a sophisticated metabolism mediated entirely by RNA would have been necessary for ribozymes to give rise to encoded protein synthesis-a precursor to the complex system of translation that is present in all extant organisms. To achieve maximum catalytic rate enhancements, natural ribozymes require Mg 2ϩ or other metal ion cofactors to induce structure formation or to participate directly in the catalytic process (4), perhaps defining a limitation to the catalytic potential for polynucleotide-based enzymes. When the limited catalytic repertoire of natural ribozymes is considered, the notion that complex networks of metabolic reactions could be guided entirely by RNA appears tenuous. Ancient ribozymes, however, may have created a larger catalytic repertoire by using various divalent metals (5) and by using nucleotide-like cofactors (6-8) to supplement the limited set of chemical groups that make up unmodified RNA. In recent years, a surprising number of artificial ribozymes have been created by using in vitro selection (9) that catalyze various chemical reactions including alkylation, acylation, phosphorylation, and ester and amide bond formation. In addition, the catalytic rates of ribozymes can be tightly regulated by using allosteric mechanisms (10), indicating that the potential for extensive and sophisticated catalysis by nucleic acids is large. Moreover, the full range of reac...

A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

“…In a previous study, it was determined that a variant of the HD3 deoxyribozyme exhibits an activity profile wherein catalytic activity increases with increasing pH up to the point at which the pH matches the pK a for the imidazole group of the cofactor, either histidine or its methyl ester (Roth and Breaker 1998). The simplest explanation for this pH profile is that the deoxyribozyme uses the cofactor for general base catalysis (Fig.…”

“…We began by conducting a succession of three assays using a histidine-dependent deoxyribozyme, termed HD3 (Roth and Breaker 1998), to define conditions under which to measure the maximum k obs . This is expected to be possible when the substrate is saturated with enzyme, the ionic strength requirements are satisfied, and the cofactor is present in sufficient quantity to saturate the enzyme-substrate complex ( Fig.…”

A Silver DNAzyme

“…[25][26][27] For example, the Lu group reported a DNAzyme with a rate of ~0.1 min -1 using Na + as the sole metal. 27 To reach such a high rate, however, 400 mM Na + is needed.…”

Riboswitch effectors as protein enzyme cofactors

“…A histidine-dependent DNAzyme was selected that is able to cleave an RNA substrate (Roth and Breaker 1998). A ribozyme that uses nicotinamide adenine dinucleotide (NAD) to catalyze redox chemistry in the presence of zinc has also been selected (Tsukiji et al 2003(Tsukiji et al , 2004).…”