Kinetic and equilibrium studies of the ribonuclease-catalyzed hydrolysis of uridine 2',3'-cyclic phosphate

Ej, Del Rosario; Gg, Hammes

doi:10.1021/bi00833a017

Cited by 67 publications

(45 citation statements)

References 21 publications

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“…1). We found RNase A to have a pH optimum of 6.0, a value that closely reflects classic studies (68) and makes RNase A well suited for the acidic environment of the bovine rumen. Conversely, human RNase 1 and BRB had pH optimums of 7.3 and 7.4, respectively, which are close to the pH of many bodily fluids, including blood (pH 7.365).…”

Section: Discussionsupporting

confidence: 78%

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

Eller

Lomax

Raines

2014

Journal of Biological Chemistry

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Background: RNase 1 is an RNA-degrading enzyme conserved in mammals and with unknown biological function. Results: RNase 1 homologs in human and cow display different biochemical and biological properties, implying divergent physiology. Conclusion: Bovine brain ribonuclease, not RNase A, is the functional homolog of human RNase 1. Significance: Fundamental insight into the biology and evolution of human RNase 1 is attained from analyses of homologous proteins.

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Section: Discussionsupporting

confidence: 78%

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

Eller

Lomax

Raines

2014

Journal of Biological Chemistry

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“…The initial rate of formation of 3′ CMP with a molecular mass of 324 Da equals V max because [S] ӷ Km. The kcat for the RNase A-catalysed cyclization of 3′ CMP equals 0.00066 s Ϫ1 , comparable with that for 3′ UMP, which was derived from the equilibrium constant for the hydrolysis reaction [6].…”

Section: % (V/v) Eg Alcoholysis Hydrolysissupporting

confidence: 55%

“…The equilibrium constant between 2′,3′ cGMP and the acyclic compounds is close to 1 M, whether the hydrolysis or alcoholysis reaction is considered. The equilibrium constants for the hydrolysis of 2′,3′ cCMP and 2′,3′ cUMP have been determined previously and equal 0.15 M and 0.13 M at pH 5.0, 25°C [5,6]. The first-order rate constant for the conversion of 3′ GMP to 2′,3′ cGMP by RNase T1 amounts to 0.00101 s Ϫ1 .…”

Section: % (V/v) Eg Alcoholysis Hydrolysismentioning

confidence: 99%

“…From the uncatalysed rates of UpA cleavage [23] and dimethyl phosphate (as a model for DNA) hydrolysis [24], we estimate the rate at which RNA depolymerizes in water to be at least 5ϫ10 6 times greater than that of DNA. In the case of RNA depolymerization, formation of the 2′,3′ cyclophosphate is unfavourable because of strain [25] and/or aqueous solvation effects [26].…”

Section: % (V/v) Eg Alcoholysis Hydrolysismentioning

confidence: 99%

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Reconsidering the energetics of ribonuclease catalysed RNA hydrolysis

Loverix

Laus

Martins

et al. 1998

European Journal of Biochemistry

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In principle, all biochemical reactions are reversible, though some are more reversible than others. The classical ribonuclease mechanism involves a reversible transphosphorylation step, followed by quasi irreversible hydrolysis of the cyclic intermediate. We performed isotope-exchange and intermediate-trapping experiments showing that the second hydrolysis step is readily reversible in the presence of RNase A or RNase T1. As a consequence, the equilibrium between a phosphodiester and a 2′,3′-cyclophosphate accounts for all catalysed reactions, even if the leaving/attacking group is a water molecule. Therefore, ribonucleases are transferases rather than hydrolases. The equilibrium constant for the catalysed interconversion is close to 1 M. From this result, we estimate the effective concentration of the 2′-hydroxyl nucleophile in the cyclization step to be 10 7 M. The high effective concentration of the vicinal hydroxyl group balances the strain-associated and solvation-associated instability of the pentacyclic phosphodiester.Keywords : ribonuclease; hydrolysis; transphosphorylation; energetics.Fission of the phosphodiester linkage is of central biochemical importance. Ribonucleases catalyse the hydrolysis of P-O5′ phosphodiester bonds of single-stranded RNA. The classical mechanism involves a reversible transphosphorylation reaction yielding a 2′,3′-cyclophosphate ( Fig. 1 A). In a second, separate step, this cyclic product is hydrolysed to yield a free 3′ phosphate [1,2]. Both catalysed reactions consist of associative nucleophilic displacements at the phosphorus. The reactions follow a concerted in-line mechanism with a trigonal bipyramidal transition state, implying a base and an acid located on either side of the scissile bond [3,4].In principle, all chemical reactions are reversible. Biochemists simply adopt a convenient nomenclature of 'irreversible' versus 'reversible', based on quantitative differences in the energetics of the forward and reverse reactions. Most authors consider the ribonuclease-catalysed hydrolysis step to be quasi irreversible. Older literature, however, indicates that 2′,3′-cyclic nucleotides can be synthesized from 3′ nucleotides by RNase A [5,6]. In the present study, we reanalysed the reversibility of the ribonuclease-catalysed hydrolysis step qualitatively and quantitatively, using isotope-exchange and intermediate-trapping experiments. RNase T1, a guanosine-specific ribonuclease [7] is the best-known representative of a homologous family of microbial ribonucleases, with members in the prokaryotic and eukaryotic world [8,9]. RNase A is the most studied member of an- other large superfamily of mammalian RNases [10]. Although RNase A and RNase T1 are not homologous in sequence or in structure, both enzymes evolved to catalyse the same chemistry. The present study reveals new aspects of the energetics of ribonuclease catalysis. MATERIALS AND METHODS Chemicals and enzymes. H 218 O, 3′ GMP, 3′ CMP, ethylene glycol (EG), and RNase A were purchased from Sigma. Recombinant wild-type RNas...

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“…5, resulting in a bell-shaped pH-rate profile. Such bell-shaped pH-rate profiles are common for imidazolecatalyzed hydrolysis of RNA (39,40) and RNase A-catalyzed hydrolysis of RNA (41,42), in that the maximal activity can be found around pH 7, reflecting the pK a of the catalytic molecules (imidazole or histidine). In the case of ribozyme-catalyzed reactions, the activity increases linearly with pH from pH 6 to up to pH 9 (13,19) in accord with Eq.…”

mentioning

confidence: 97%

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

Zhou

Zhang

Taira

1997

Proc. Natl. Acad. Sci. U.S.A.

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In a previous examination using natural all-RNA substrates that contained either a 5-oxy or 5-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5-thio group (R11S) and (ii) the departure of the 5 leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg 2؉ ions were replaced by Mn 2؉ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg 2؉ ions were replaced by Mn 2؉ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5-oxygen, because a switch in metal ion specificity was not observed with the 5-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis.Among various catalytic RNAs, the hammerhead-type ribozyme is the smallest and best understood as far as the relationship between structure and function is concerned. Naturally occurring hammerhead ribozymes can be found in some RNA viruses, and they act ''in cis'' during viral replication by the rolling circle mechanism (1-3). The hammerhead ribozyme has been engineered in such a way that it can act ''in trans'' against other RNA molecules (4, 5). The trans-acting hammerhead ribozyme developed by Haseloff and Gerlach (5) consists of an antisense section (stems I and III) and a catalytic domain with a flanking stem II and loop section (Fig. 1a). Because of the small size of hammerhead ribozymes, they are well suited for mechanistic studies, being good representatives of catalytic RNAs. Recently, hammerhead ribozymes were crystallized and their structures were analyzed in detail by two independent groups (6-9). The global three-dimensional structures of the two crystallized ribozymes were nearly identical: one domain of the conserved core, which consists of the sequence C 3 U and is located next to stem I, makes a sharp turn identical to the uridine turn in tRNAs (10). As a result, stem II and stem III are aligned almost colinearly through pseudocontinuous, long A-type helices.It is now well established that ribozymes are metalloenzymes (2,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Although x-ray analysis of a hammerhead ribozyme identified one potential catalytic metal ion (7-9), the exact number of metal ions required for catalysis remains obscure, because the newly captured conformational intermediate appears to demand further conformational change for the following in-line attack (9). Direct evidence that a Mg 2ϩ ion acts as a Lewis acid by coordinating directly to the leaving ...

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Kinetic and equilibrium studies of the ribonuclease-catalyzed hydrolysis of uridine 2',3'-cyclic phosphate

Cited by 67 publications

References 21 publications

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

Reconsidering the energetics of ribonuclease catalysed RNA hydrolysis

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

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