Stability constants of magnesium and cadmium complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of magnesium-ATP and magnesium-ADP

Abstract

Stability constants for the Mg2+ and Cd2+ complexes of ATP, ADP, ATP alpha S, ATP beta S, and ADP alpha S have been determined at 30 degrees C and mu = 0.1 M by 31P NMR. Besides being of the utmost importance for determining species distributions for enzymatic studies, these constants allow an estimation of the preference of Cd2+ for sulfur vs. oxygen coordination in phosphorothioate complexes. Stability constants for Mg2+ complexes decreases when sulfur replaces oxygen (log K: ADP, 4.11; ADP alpha S, 3.66; ATP, 4.70; ATP alpha S, 4.47; ATP beta S, 4.04) because of (a) a statistical factor resulting from the loss of one potential phosphate oxygen ligand and (b) either an alteration in the charge distribution between oxygen and sulfur or destabilization of the chelate ring structure by loss of an internal hydrogen bond between an oxygen of coordinated phosphate and metal-bound water. Cd2+ complexes with sulfur-substituted nucleotides are more stable than those without sulfur (log K: ADP, 3.58; ADP alpha S, 4.95; ATP, 4.36; ATP alpha S, 4.42; ATP beta S, 5.44) because of the preferential binding of Cd2+ to sulfur rather than oxygen, which we estimate to be approximately 60 in CdADP alpha S and CdATP beta S. The proportion of tridentate coordination is estimated to be 50-60% in MgATP and MgATP beta S, approximately 27% in MgATP alpha S, approximately 16% in CdATP or CdATP beta S, but approximately 75% in CdATP alpha S. By analysis of the data of Jaffe and Cohn [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839], we conclude that the preference for oxygen over sulfur coordination to ATP beta S is 31 000 for Mg2+, 3100-3900 for Ca2+, and 158-193 for Mn2+. Proton NMR demonstrates that bidentate Cd2+ complexes form intramolecular chelates with the N-7 of adenine while Mg2+ nucleotides and the tridenate CdATP alpha S do not. An analysis of the 31P NMR line widths shows that the rate constants for dissociation of MgADP and MgATP are both 7000 s-1 while the association rate constants are 7 X 10(7) and 4 X 10(8) M-1 s-1, respectively. The observed dependence of the line width on nucleotide concentration is best explained by a base-stacking model at nucleotide concentrations above 5 mM.

Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism

“…Preparative-scale processing by chloroplast RNase P of phosphorothioate-substituted chloroplast pre-tRNA Phe + Precursor tRNAs were either unsubstituted or were uniformly substituted with GMPaS and were labeled singly with [ 32 P]UMP, [ 32 P] AMP, or [ 32 P]CMP+ Each 100-mL reaction contained ;2 U HA-Ultrogel-purified RNase P and ;5 nM maize chloroplast pre-tRNA Phe + The reaction was incubated at 37 8C for 45 min; products were separated by electrophoresis and detected by autoradiography+ Metal coordination at the scissile pro-R P oxygen is unlikely to be rate limiting for chloroplast RNase P To further investigate the mechanism whereby uniform phosphorothioate substitution reduces cleavage velocity, we determined Michaelis-Menten steady-state kinetic constants+ We expect that an inhibition resulting from decreased Mg 2ϩ coordination will reduce the catalytic rate constant, k chem , by an amount commensurate with the decreased affinity of Mg 2ϩ for sulfur versus for oxygen, or about 10 3 -to 10 4 -fold if the catalytic step is the rate-limiting component of k cat (Pecoraro et al+, 1984; discussed in Chen et al+, 1997)+ Even if catalysis is not rate limiting, thiosubstitution will produce a profound inhibition of multiple-turnover reactions (cf+ Chen et al+, 1997)+ On the other hand, if there is no metal coordination by the pro-R P oxygen, we expect an extent of inhibition commensurate with that of the reduced reactivity of sulfur-linked phosphorus, which has been measured at 4-to 11-fold for model reactions (Herschlag et al+, 1991)+ To this end we compared substrate dependence in kinetic assays using unsubstituted chloroplast pre-tRNA Phe and GMPaS-containing pre-tRNA Phe + The kinetic constants obtained from the best-fitting Michaelis-Menten curves are summarized in Table 3+ Unsubstituted pre-tRNA Phe is cleaved by chloroplast RNase P with a V max of 2+7 nM 59 leader formed min Ϫ1 + The observed K M , 16 nM pre-tRNA, is similar to that for bacterial RNase P (Stark et al+, 1978; FIGURE 4. Determination of RNase P cleavage site in chloroplast pre-tRNA Phe + The tRNA ϩ 39 trailer products from a preparative-scale processing reaction (shown in Fig+ 3) were digested with RNase T2+ The resulting nucleotides were separated on PEI-cellulose thin-layer plates developed with 1+6 M LiCl and were detected by autoradiography+ The migration positions of unlabeled pAp and pGp, added as internal standards, are indicated by dotted lines+ The identity of p (S) Gp was determined as described in Materials and Methods+ Substrate titrations were performed using pre-tRNAs that were either unsubstituted or 100% substituted with GMPaS+ Each 20-mL reaction contained 0+1 U of HA-Ultrogel-purified RNase P and increasing amounts of precursor tRNA+ Reactions were performed and analyzed as described in Materials and Methods+ McClain et al+, 1987;Reich et al+, 1988;Smith & Pace, 1993)+ In comparison, for pre-tRNA Phe uniformly substituted with GMPaS, V max is reduced about sevenfold, to 0+38 nM 59 leader formed min Ϫ1 , although K M remains almost unchanged at 12 nM pre-tRNA+ The overall throughput of chloroplast RNase P in processing these two pre-tRNAs was estimated from the value of V max /K M , which, as indicated in Table 3, decreases fivefold upon phosphorothioate substitution+ Chloroplast RNase P is hence fivefold less efficient when processing a phosphorothioate-substituted pre-tRNA than an unsubstituted prec...…”

Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P

“…Processing of thiosubstituted pre-tRNA Phe by S. cerevisiae nuclear RNase P+ Substrates pre-G 1 Phe (Base 1: G) or pre-A 1 Phe (Base 1: A) were unsubstituted (Thio: Ϫ) or substituted with either AMPaS (Thio: A) or GMPaS (Thio: G)+ pre: pre-tRNA; 59: 59-leader; *: products of aberrant cleavage as discussed in the text+ A: Failure to cleave phosphorothioate bonds+ Processing reactions were performed for 30 min at 30 8C with 0+1 pmol pre-tRNA Phe -G 1 (lanes 1-3, 7-9) or pre-tRNA Phe -A 1 (lanes 4-6, 10-12), plus 1 mL of yeast nuclear RNase P (lanes 1-6) or no enzyme (lanes 7-12)+ B: Mn(II) does not restore cleavage at phosphorothioate bonds+ Substrates pre-G 1 Phe or pre-A 1 Phe were unsubstituted or substituted with AMPaS or GMPaS as indicated+ Reactions were performed with 1 mL RNase P (lanes 1-18) or no enzyme (lanes 19-20) for 30 min at 30 8C in buffer containing MgCl 2 or MnCl 2 at the concentrations indicated+ Reactions in lanes 19-20 contained 8 mM MgCl 2 + raro et al+, 1984;Piccirilli et al+, 1993;Warnecke et al+, 1996;Chen et al+, 1997)+ We attempted to restore activity to thio-inhibited reactions by addition of 5 mm MnCl 2 to reactions containing 8 mm MgCl 2 , as shown in Figure 2B+ Cleavage of pre-G 1 Phe and pre-A 1 Phe, unsubstituted or substituted with AMPaS or GMPaS, was first repeated in the presence of 8 mM MgCl 2 only (Fig+ 2B, lanes 1-6)+ In those reactions in which the thiosubstitution occurs at the scissile bond (Fig+ 2B, lanes 3 and 5), the normal RNase P cleavage products do not appear, but are replaced by small amounts of species migrating more slowly than tRNA and more quickly than 59 leader+ The size of the smaller RNA suggests that these species could be generated by a single cleavage 1-2 nt upstream of the normal tRNA 59 terminus+ When the same reactions are performed in the presence of 5 mM MnCl 2 , the extent of processing is substantially reduced, but the appearance of incorrectly processed products (Fig+ 2B, lanes 9 and 11) is not altered+ Likewise, an identical pattern is seen in reactions containing both 8 mM MgCl 2 and 5 mM MnCl 2 (Fig+ 2B, lanes 13-18)+ The incorrect cleavage of substrates with phosphorothioate scissile bonds is not detectably corrected under these conditions (Fig+ 2B, lanes 15 and 17)+ The extent of cleavage and the sizes of RNAs detected after RNase P reaction of pre-tRNAs containing thiosubstituted scissile bonds (Fig+ 2B,lanes 3,5,9,11,15,17) are similar to those observed after the incubation of unsubstituted substrates in the absence of enzyme (Fig+ 2B,lanes 19 and 20), suggesting that the aberrant RNA species are breakdown products not dependent upon RNase P+…”

Metal ion-N7 coordination in a ribozyme branch domain by NMR

“…[4][5][6] In larger RNA structures, the situation is more complex, as aside from metal ion binding, also the binding mode, e.g., inner versus outer sphere, as well as small structural changes affect the proton chemical shifts. Metal ion coordination to bridging phosphate moieties has been probed by thiophosphate modification, [7][8][9][10][11][12] but for the nucleobase residues the situation is more complicated. The N7 of purine bases is thereby of special interest as this atom constitutes a prominent coordination site for metal ions in nucleic acids.…”

“…11 This effect can be attributed to the fast on-and off-rates of first-shell ligandexchange of Mg 2+ , which is in the order of the NMR time scale. The substantial line broadening effect observed at A14N7, which is located in the tetraloop, could thus be a direct consequence of inner-sphere binding to this site.…”