Transphosphorylation catalyzed by ribonuclease A: computational study using ab initio effective fragment potentials

Wladkowski, Brian D.; Krauß, M.; Stevens, Walter J.

doi:10.1021/ja00147a015

Cited by 45 publications

(74 citation statements)

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“…27 This analysis is extended in Figure 4, which is a plot of logK p vs log(K m /k cat ). The shallow slope of this plot (near 0.2) indicates that Lys41 interacts far more extensively with the transition state than the product during catalysis of C>p hydrolysis.…”

Section: Is Uridine 2′3′-cyclic Vanadate a Transition State Analogue?mentioning

confidence: 98%

“…[27][28][29] The Cys41 sulfhydryl group of K41C RNase A was modified by alkylation with bromoethylamine to yield a semisynthetic variant with an S-(aminoethyl)cysteine residue at position 41 (K41CEA RNase A) as described. 27 Uridine 3′-(p-nitrophenyl phosphate) [Up(OC 6 H 4 -p-NO 2 )] was synthesized by J. E. Thompson and T. G. Kutateladze as the 2′,5′-O-tetrahydropyranyl-protected material,30 and handled as described. 31 Sodium vanadate was obtained from Aldrich Chemical (Milwaukee, WI).…”

Section: Experimental Section Materialsmentioning

confidence: 99%

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Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Messmore

Raines

2000

J. Am. Chem. Soc.

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Pentavalent organo-vanadates have been put forth as transition state analogues for a variety of phosphoryl transfer reactions. In particular, uridine 2′,3′-cyclic vanadate (U>v) has been proposed to resemble the transition state during catalysis by ribonuclease A (RNase A). Here, this hypothesis is tested. Lys41 of RNase A is known to donate a hydrogen bond to a nonbridging phosphoryl oxygen in the transition state during catalysis. Site-directed mutagenesis and semisynthesis were used to create enzymes with natural and nonnatural amino acid residues at position 41. These variants differ by 10 5 -fold in their k cat /K m values for catalysis, but <40-fold in their K i values for inhibition of catalysis by U>v. Plots of logK i vs log(K m /k cat ) for three distinct substrates [poly(cytidylic acid), uridine 3′-(p-nitrophenyl phosphate), and cytidine 2′,3′-cyclic phosphate] have slopes that range from 0.25 and 0.36. These plots would have a slope of unity if U>v were a perfect transition state analogue. Values of K i for U>v correlate weakly with the equilibrium dissociation constant for the enzymic complexes with substrate or product, indicating that U>v bears some resemblance to the substrate and product as well as the transition state. Thus, U>v is a transition state analogue for RNase A, but only a marginal one. This finding indicates that a pentavalent organo-vanadate cannot necessarily be the basis for a rigorous analysis of the transition state for a phosphoryl transfer reaction.

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Section: Is Uridine 2′3′-cyclic Vanadate a Transition State Analogue?mentioning

confidence: 98%

Section: Experimental Section Materialsmentioning

confidence: 99%

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Messmore

Raines

2000

J. Am. Chem. Soc.

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“…This then expels the 5′-hydroxyl group of the second nucleotide to cleave the P-O bond in a second step. We proposed this two-step phosphorane process to be the preferred path for the enzyme, a proposal further supported by computation (9)(10)(11). For a general review of phosphoranes as well-demonstrated intermediates in phosphate reactions, see ref.…”

mentioning

confidence: 88%

Deoxypolypeptides bind and cleave RNA

Cheng

Mahendran

Gonzalez

et al. 2014

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

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We have prepared L-and D-deoxypolypeptides (DOPPs) by selective reduction of appropriately protected polyhistidines with borane, reducing the carbonyl groups to methylenes. The result is a chiral polyamine, not amide, with a mainly protonated backbone and chirally mounted imidazolylmethylene side chains that are mostly unprotonated at neutrality because of the nearby polycationic backbone. We found that, in contrast with the D-octahistidine DOPP, the L-octahistidine DOPP is able to cooperatively bind to a D-polyuridylic acid RNA; this is consistent with results of previous studies showing that, relative to D-histidine, L-histidine is able to more strongly bind to RNA. The L-DOPP was also a better catalyst for cleaving the RNA than the D-DOPP, consistent with evidence that the L-DOPP uses its imidazole groups for catalysis, in addition to the backbone cations, but the D-DOPP does not use the imidazoles. The L-DOPP bifunctional process probably forms a phosphorane intermediate. This is a mechanism we have proposed for models of ribonuclease cleavage and for the ribonuclease A enzyme itself, based on our studies of the cleavage and isomerization of UpU catalyzed by imidazole buffers as well as other relevant studies. This mechanism contrasts with earlier, generally accepted ribonuclease cleavage mechanisms where the proton donor coordinates with the oxygen of the leaving group as the 2-hydroxyl of ribose attacks the unprotonated phosphate. T he enzyme ribonuclease A (RNase A) hydrolyzes RNA with bifunctional catalysis, using His-12 as a base and protonated His-119 as an acid to form the first products, a 2,3-cyclic phosphate of one ribose and the free next ribose with a new terminal 5′-hydroxyl group (1, 2). Lys-41 is also involved in stabilizing intermediate anions (3). In a second step the cyclic phosphate is hydrolyzed using the now-protonated His-12 and now-deprotonated His-119 in essentially a reverse of the cyclization step, with water replacing the second ribose unit (4). In the classical mechanism of this process it had been generally accepted, including in textbooks, that the protonated His-119 donates its proton to the leaving group whereas the 2′-hydroxyl group attacks the phosphate (1, 2) [isotope studies show that two protons are "in flight" during the hydrolysis step, essentially the reverse of the cyclization, both moving at the same time (5)]. We also showed the two-proton-in-flight process in a model system (6).However, our extensive studies on the cleavage of RNA with imidazole-imidazolium catalysts and on catalysis by RNase A itself indicated that a different mechanism is used ( Fig. 1) (7, 8). The proton donor BH + hydrogen binds to a phosphate oxyanion and donates the proton to the phosphate as the ribose 2′-hydroxyl adds while losing a proton to base :B. This process results in an intermediate phosphorane species. This then expels the 5′-hydroxyl group of the second nucleotide to cleave the P-O bond in a second step. We proposed this two-step phosphorane process to be the preferred path for th...

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“…According to the generally accepted mechanism [1,27] His12 was unprotonated, and Lys41 and His119 were protonated. In addition, to evaluate our docking method, we also used the RNase A -cyclic uridyl phosphate (CUP) complex from previously published QM/MM calculations [28] to perform docking studies between CUP and the protein to reproduce this structure for verification.…”

Section: Protein Preparation In Silicomentioning

confidence: 99%

Nucleotide docking: prediction of reactant state complexes for ribonuclease enzymes

Elsässer

Fels

2010

J Mol Model

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International audienceRibonuclease enzymes (RNases) play key roles in the maturation and metabolism of all RNA molecules. Computational simulations of the processes involved can help to elucidate the underlying enzymatic mechanism and is often employed in a synergistic approach together with biochemical experiments. Theoretical calculations require atomistic details regarding the starting geometries of the molecules involved, which, in the absence of crystallographic data, can only be achieved from computational docking studies. Fortunately, docking algorithms have improved tremendously in recent years, so that reliable structures of enzyme-ligand complexes can now be successfully obtained from computation. However, most docking programs are not particularly optimized for nucleotide docking. In order to assist our studies on the cleavage of RNA by the two most important ribonuclease enzymes, RNase A and RNase H, we evaluated four docking tools--MOE2009, Glide 5.5, QXP-Flo+0802, and Autodock 4.0--for their ability to simulate complexes between these enzymes and RNA oligomers. To validate our results, we analyzed the docking results with respect to the known key interactions between the protein and the nucleotide. In addition, we compared the predicted complexes with X-ray structures of the mutated enzyme as well as with structures obtained from previous calculations. In this manner, we were able to prepare the desired reaction state complex so that it could be used as the starting structure for further DFT/B3LYP QM/MM reaction mechanism studies

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Transphosphorylation catalyzed by ribonuclease A: computational study using ab initio effective fragment potentials

Cited by 45 publications

References 0 publications

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Deoxypolypeptides bind and cleave RNA

Nucleotide docking: prediction of reactant state complexes for ribonuclease enzymes

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