Structure (1.3 Å) and Charge States of a Ribonuclease A−Uridine Vanadate Complex:  Implications for the Phosphate Ester Hydrolysis Mechanism

Wladkowski, Brian D.; Svensson, L.A.; Sjölin, L.; Ladner, Jane E.; Gilliland, Gary L.

doi:10.1021/ja972296n

Cited by 45 publications

(71 citation statements)

References 69 publications

(107 reference statements)

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“…4! closely resemble those previously reported for complexes of RNase A with uridine-containing nucleotides Fisher et al, 1998;Wladkwosky et al, 1998!. Uridine O2 and N3 form strong hydrogen bonds with N and O g1 of Thr45, respectively.…”

Section: Detailed Description Of the Active Sites Of Molecules I And Iisupporting

confidence: 87%

“…The glycoside linkage between the uridine base and the ribose is anti-and the pseudo-rotation phase angle shows that the sugar ring adopts a C39-endo puckering. Similar ring puckerings have been reported in the productive bindings of the transition state analog uridine~29, 39!cyclic vanadate~Wlodawer et al, 1983;Wladkwosky et al, 1998! andof cytidylic acid~Lisgarten et al, 1993!. In molecule II a cluster of water molecules is located in the active site.…”

Section: Detailed Description Of the Active Sites Of Molecules I And Iisupporting

confidence: 67%

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Productive and nonproductive binding to ribonuclease A: X‐ray structure of two complexes with uridylyl(2′,5′)guanosine

et al. 2000

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Guanine-containing mono-and dinucleotides bind to the active site of ribonuclease A in a nonproductive modẽ retro-binding!~Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA, 1992, J Mol Biol 224:265-267!. Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro-binding, we determined the structure of two new complexes of RNase A with uridylyl~29,59!guanosine obtained by soaking two different forms of pre-grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X-ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 Å~R-factor 0.192! and 2.0 Å~R-factor 0.178!, respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro-binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand-bound and a ligand-free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site-directed mutagenesis studies.

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Section: Detailed Description Of the Active Sites Of Molecules I And Iisupporting

confidence: 87%

Section: Detailed Description Of the Active Sites Of Molecules I And Iisupporting

confidence: 67%

Productive and nonproductive binding to ribonuclease A: X‐ray structure of two complexes with uridylyl(2′,5′)guanosine

et al. 2000

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“…In the crystalline RNase A•U>v complex, active-site residues are found in positions not congruent with their commonly accepted mechanistic roles. [22][23][24] For example, we have used semisynthesis to show that Lys41 donates a hydrogen bond to a non-bridging phosphoryl oxygen in the transition state for RNA cleavage. 25 Yet, the side-chain amino group of Lys41 is much closer to the 2′ oxygen than to a non-bridging vanadyl oxygen in the RNase A•U>v complex ( Figure 1C).…”

Section: Introductionmentioning

confidence: 99%

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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“…The lack of large thioeffects for the RNase A-catalyzed reaction (11) argues against important catalytic interactions with the nonbridging oxygens. Moreover, crystallographic studies of RNase A complexed with a 3Ј-mononucleotide substrate (3Ј-CMP (12) and 3Ј-UMP (13)) or with the transition state analog uridine vanadate (14) persistently show a short hydrogen bond of about 2.7 Å between N of Lys-41 and the 2Ј-oxygen, much like His-40 in RNase T 1 (Fig. 2c).…”

mentioning

confidence: 99%

A Nucleophile Activation Dyad in Ribonucleases

Mignon¹,

Steyaert²,

Loris³

et al. 2002

Journal of Biological Chemistry

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Ribonucleases (RNases) catalyze the cleavage of the phosphodiester bond in RNA up to 10 15 -fold, as compared with the uncatalyzed reaction. High resolution crystal structures of these enzymes in complex with 3-mononucleotide substrates demonstrate the accommodation of the nucleophilic 2-OH group in a binding pocket comprising the catalytic base (glutamate or histidine) and a charged hydrogen bond donor (lysine or histidine). Ab initio quantum chemical calculations performed on such Michaelis complexes of the mammalian RNase A (EC 3.1.27.5) and the microbial RNase T 1 (EC 3.1.27.3) show negative charge build up on the 2-oxygen upon substrate binding. The increased nucleophilicity results from stronger hydrogen bonding to the catalytic base, which is mediated by a hydrogen bond from the charged donor. This hitherto unrecognized catalytic dyad in ribonucleases constitutes a general mechanism for nucleophile activation in both enzymic and RNAcatalyzed phosphoryl transfer reactions.RNases have been the subject of landmark research in areas ranging from basic protein chemistry to enzymology to protein folding and crystallography. These enzymes catalyze the intramolecular nucleophilic displacement of the 5Ј-leaving group by the attacking 2Ј-hydroxyl group in RNA, forming a 2Ј,3Ј-cyclophosphate (see Fig. 1). The nucleophilic attack occurs inline, in a postulated trigonal bipyramidal transition state, implying a catalytic base and acid on either side of the scissile bond (1, 2). X-ray crystallographical and site-directed mutagenesis experiments have provided substantial insight in the structure-function relationship of two unrelated families of ribonucleases. RNase A, (bovine pancreatic ribonuclease A) (EC 3.1.27.5) (3) and RNase T 1 , (EC 3.1.27.3) (4, 5) (and references therein) are the best characterized members of the mammalian and the microbial enzymes, respectively. The mammalian enzymes depend on two histidines for acid/base catalysis, whereas a histidine/glutamic acid pair is found in the microbial enzymes (6).The active site of RNase T 1 is composed of the side chains of Tyr-38, His-40, Glu-58, Arg-77, His-92, and Phe-100 (see Fig. 2a). With the exception of Arg-77, all these amino acids have been shown to take part in catalysis (5). Both the His-40 and Glu-58 side chains are in the direct vicinity of the 2Ј-nucleophile. pH dependence studies have shown Glu-58 to be unprotonated and His-40 to be protonated at the onset of catalysis, proving that Glu-58 is the catalytic base accepting a proton from the 2Ј-nucleophile (7). The protonated His-92 located at the opposite side of the active site functions as the catalytic acid. Removal of the His-40 side chain leads to a 6500-fold decrease in the second-order rate constant k cat /K m suggesting an important catalytic role for this residue in the wild type enzyme. His-40 is believed to polarize the 2Ј-hydroxyl group and to properly orientate Glu-58 toward the substrate (8, 9). However, the actual role of this residue is not totally clarified at present. Despite the l...

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Structure (1.3 Å) and Charge States of a Ribonuclease A−Uridine Vanadate Complex: Implications for the Phosphate Ester Hydrolysis Mechanism

Cited by 45 publications

References 69 publications

Productive and nonproductive binding to ribonuclease A: X‐ray structure of two complexes with uridylyl(2′,5′)guanosine

Productive and nonproductive binding to ribonuclease A: X‐ray structure of two complexes with uridylyl(2′,5′)guanosine

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

A Nucleophile Activation Dyad in Ribonucleases

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