NMR studies of ion binding to Escherichia coli tRNAPhe

The effects of magnesium, spermine, and temperature on the conformation of Escherichia coli tRNAPhe have been examined by proton and phosphorus nuclear magnetic resonance spectroscopy. In the low-field proton NMR spectra we have characterized two slowly interconverting conformations of this tRNA at low magnesium ion concentrations. The relative proportion of the conformers is ion dependent but not ion specific. Magnesium affects protons in all the stems of tRNA while spermine effects are localized near the s4U-8-A-14 and G-15-C-48 tertiary bonds. The effects seen in the proton NMR spectra are compared and correlated with those observed in the phosphorus spectra to give assignments of some of the resolved signals from the phosphate groups. The phosphorus spectra are compared with those of yeast tRNAPhe [Gorenstein, D. G., Goldfield, E. M., Chen, R., Kovar, K., & Luxon, B. A. (1981) Biochemistry 20, 2141; Salemink, P. J. M., Reijerse, E. J., Mollevanger, L., & Hilbers, C. W. (1981) Eur. J. Biochem. 115, 635], and the ion effects are discussed with reference to the magnesium and spermine sites found in the crystal structures of yeast tRNAPhe [Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, S.-H. (1977) Nucleic Acids Res. 4, 2811; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64; Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315].     

Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid.

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.     

Phosphorus-31 nuclear magnetic resonance studies of the conformation of an adenosine 5'-triphosphate analogue at the active site of (Na+ + K+)-ATPase from kidney medulla

It has previously been shown that there are two sites for divalent metals at the active site of kidney (Na+ + K+)-ATPase, one bound directly to the enzyme and one coordinated to the ATP substrate [Grisham, C. (1981) J. Inorg. Biochem. 14, 45; O'Connor, S., & Grisham, C. (1980) FEBS Lett. 118, 303]. The conformation of the metal-nucleotide complex has been studied by using beta, gamma-bidentate Co-(NH3)4ATP, a substitution-inert analogue of MgATP. Kinetic studies show that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the (Na+ + K+)-ATPase. The Ki values under both high- and low-affinity conditions (Ki = 10 microM and Ki = 1.6 mM, respectively) are similar to the Km values for MnATP under the same conditions (2.88 microM and 0.902 mM). From the paramagnetic effect of Mn2+ bound to the ATPase on the longitudinal relaxation rates of the phosphorus nuclei of Co(NH3)4ATP at the substrate site (at 40.5 and 145.75 MHz), Mn-P distances to all three phosphates are determined. The distances are consistent with the formation of a second sphere coordination complex on the enzyme between Mn2+ and the phosphates of Co(NH3)4ATP. In this respect, kidney (Na+ + K+)-ATPase appears to be similar to pyruvate kinase [Sloan, D., & Mildvan, A. (1976) J. Biol. Chem. 251, 2412] and phosphoribosylpyrophosphate synthetase [Granot, J., Gibson, K., Switzer, R., & Mildvan, A. (1980) J. Biol. Chem. 255, 10931]. Roles for both of the active site divalent cations are discussed.     

Analysis of the high- and low-spin Soret bands of horse-heart metmyoglobin complexes

Interest in the thermodynamics of the iron-binding site in hemoproteins has increased in recent years due to refinements in x-ray crystallographic studies of hemoproteins [see Deathage, J. F., Lee, R. S., Anderson, C. M. & Moffat, K. (1976) J. Mol. Biol. 104 , 687–706; Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976) J. Mol. Biol. 104 , 707–722; Deathage, J. F., Lee, R. S. & Moffat, K. (1976) J. Mol. Biol. 104 , 723–728; Ladner, R. C., Heidner, E. J. & Perutz, M. F. (1976) J. Mol. Biol. 114 , 385–414; Fermi, G. & Perutz, M. F. (1977) J. Mol. Biol. 114 , 421–431; Takano, T. (1977) J. Mol. Biol. 110 , 537–568 and 569–589], the synthesis and x-ray analysis of model heme compounds [see Scheidt, W. R. (1977) Acc. Chem. Res. 10 , 339–345; Kastner, M. E., Scheidt, W. R., Mashino, T. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 666–667; Mashiko, T., Kastner, M. E., Spartalian, K., Scheidt, W. R. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 6354–6362; Hill, H. A. O., Skite, P. P., Buchler, J. W., Luchr, H., Tonn, M., Gregson, A. K. & Pellizer, G. (1979) Chem. Commun. 4 , 151–152; and Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979) Biochemistry 18 , 3546–3556], and the numerous data on heme–protein interactions that account for the differences observed in ligand binding between the various species of animals. Numerous probes have been used and provide information about the structure and thermodynamics of the binding site, but no single probe can provide the complete picture [see Iizuka, T. & Yonetani, T. (1970) Adv. Biophys. 1 , 157–182; Smith, D. W. & Williams, R. J. P. (1970) Struct. Bond. 7 , 1–45; and Spiro, T. G. (1975) Biochim. Biophys. Acta 416 , 169–189].     

Role of divalent cations in the 3',5'-exonuclease reaction of DNA polymerase I.

X-ray studies of the proofreading 3',5'-exonuclease site of the large (Klenow) fragment of DNA polymerase I have detected a binuclear metal complex consisting of a pentacoordinate metal (site A) which shares a ligand, Asp-355, with an octahedral metal (site B) [Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., & Steitz, T. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8924-8928; Beese, L. S., & Steitz, T. A. (1991) EMBO J. 10, 25-33]. Kinetic studies of the activation of the 3',5'-exonuclease reaction by Co2+, Mn2+, or Mg2+, at low concentrations of DNA, reveal sigmoidal activation curves for the three metal ions with Hill coefficients of 2.3-2.4 and K0.5 values of 16.6 microM, 4.2 microM, and 343 microM, respectively. The binding of Co2+ to the enzyme results in the appearance of an intense visible absorption spectrum of the metal ion with maxima at 633, 570, and 524 nm and extinction coefficients of 190, 194, and 150 M-1 cm-1, respectively, suggesting the formation of a pentacoordinate Co2+ complex. Optical titration with Co2+ yields a sigmoidal titration curve which is best fit by assuming the cooperative binding of three Co2+ ions with a K0.5 of 39.9 microM, comparable to the value of 16.6 microM obtained kinetically. Displacement of Co2+ by 1 equiv of Zn2+, which binds tightly to the A site of the 3',5'-exonuclease, shifts the optical spectrum to 524 nm and lowers the extinction coefficient to 30 -1 cm-1, indicative of octahedral coordination.2+ the formation of the binuclear complex.     

Trapping of transition metal-nucleotide complexes in myosin subfragment 1 by cross-linking thiols; divalent transition metal probes of the active site

Recent experiments [Wells, J., & Yount, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966] have shown it is possible to trap MgADP and other nucleotides stably at the active site of myosin by cross-linking two thiol groups. A variety of cross-linking reagents including chelation of the two thiols by cobalt (III) phenanthroline or covalent reaction with N,N'-p-phenylenedimaleimide (pPDM) are effective trapping agents. No trapping of nucleotides occurs in the absence of divalent metals. Thus far Mg2+, Mn2+, Co2+, Ni2+, and Ca2+ but not Zn2+ all function to promote trapping of the 1:1 divalent metal-ADP complex and to enhance the rate of ATPase inactivation. Substitution-inert Cr(III) complexes of ADP, ATP, or pyrophosphate that bind very weakly or not at all to the active site are not trapped by cross-linking. While the stability of the trapped divalent metals varies, e.g., t1/2 of 0.5-7 days at 0 degree C, they are stable enough to permit accurate spectral measurements of the Mn2+ and Co2+ trapped complexes. Electron paramagnetic resonance (EPR) measurements of Mn2+ bound to 5'-adenylyl imidodiphosphate or complexed to myosin chymotryptic subfragment 1 indicate that the metal is bound at the active site. Circular dichroism (CD) and visible absorption studies of the Co2+ . ADP trapped complex indicate the metal ion is in an asymmetric octahedral environment. EPR and CD measurements show that the environment of the metal nucleotide is the same whether bound reversibly or stably trapped at the active site.     

Kinetic and magnetic resonance studies of the role of metal ions in the mechanism of Escherichia coli GDP-mannose mannosyl hydrolase, an unusual nudix enzyme

Escherichia coli GDP-mannose mannosyl hydrolase (GDPMH), a homodimer, catalyzes the hydrolysis of GDP-alpha-D-sugars to yield the beta-D-sugar and GDP by nucleophilic substitution with inversion at the C1' carbon of the sugar [Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608]. GDPMH requires a divalent cation for activity such as Mn2+ or Mg2+, which yield similar kcat values of 0.15 and 0.13 s(-1), respectively, at 22 degrees C and pH 7.5. Kinetic analysis of the Mn2+-activated enzyme yielded a K(m) of free Mn2+ of 3.9 +/- 1.3 mM when extrapolated to zero substrate concentration (K(a)Mn2+), which tightened to 0.32 +/- 0.18 mM when extrapolated to infinite substrate concentration (K(m)Mn2+). Similarly, the K(m) of the substrate extrapolated to zero Mn2+ concentration (K(S)(GDPmann) = 1.9 +/- 0.5 mM) and to infinite Mn2+ concentration (K(m)(GDPmann) = 0.16 +/- 0.09 mM) showed an order of magnitude decrease at saturating Mn2+. Such mutual tightening of metal and substrate binding suggests the formation of an enzyme-metal-substrate bridge complex. Direct Mn2+ binding studies, monitoring the concentration of free Mn2+ by EPR and of bound Mn2+ by its enhanced paramagnetic effect on the longitudinal relaxation rate of water protons (PRR), detected three Mn2+ binding sites per enzyme monomer with an average dissociation constant (K(D)) of 3.2 +/- 1.0 mM, in agreement with the kinetically determined K(a)Mn2+. The enhancement factor (epsilon(b)) of 11.5 +/- 1.2 indicates solvent access to the enzyme-bound Mn2+ ions. No cross relaxation was detected among the three bound Mn2+ ions, suggesting them to be separated by at least 10 A. Such studies also yielded a weak dissociation constant for the binary Mn2+-GDP-mannose complex (K1 = 6.5 +/- 1.0 mM) which significantly exceeded the kinetically determined K(m) values of Mn2+, indicating the true substrate to be GDP-mannose rather than its Mn2+ complex. Substrate binding monitored by changes in 1H-15N HSQC spectra yielded a dissociation constant for the binary E-GDP-mannose complex (K(S)(GDPmann)) of 4.0 +/- 0.5 mM, comparable to the kinetically determined K(S) value (1.9 +/- 0.5 mM). To clarify the metal stoichiometry at the active site, product inhibition by GDP, a potent competitive inhibitor (K(I) = 46 +/- 27 microM), was studied. Binding studies revealed a weak, binary E-GDP complex (K(D)(GDP) = 9.4 +/- 3.2 mM) which tightened approximately 500-fold in the presence of Mn2+ to yield a ternary E-Mn2+-GDP complex with a dissociation constant, K3(GDP) = 18 +/- 9 microM, which overlaps with the K(I)(GDP). The tight binding of Mn2+ to 0.7 +/- 0.2 site per enzyme subunit in the ternary E-Mn2+-GDP complex (K(A)' = 15 microM) and the tight binding of GDP to 0.8 +/- 0.1 site per enzyme subunit in the ternary E-Mg2+-GDP complex (K3 < 0.5 mM) indicate a stoichiometry close to 1:1:1 at the active site. The decrease in the enhancement factor of the ternary E-Mn2+-GDP complex (epsilon(T) = 4.9 +/- 0.4) indicates decreased solvent access to the active site Mn2+, consistent with an E-Mn2+-GDP bridge complex. Fermi contact splitting (4.3 +/- 0.2 MHz) of the phosphorus signal in the ESEEM spectrum established the formation of an inner sphere E-Mn2+-GDP complex. The number of water molecules coordinated to Mn2+ in this ternary complex was determined by ESEEM studies in D2O to be two fewer than on the average Mn2+ in the binary E-Mn2+ complexes, consistent with bidentate coordination of enzyme-bound Mn2+ by GDP. Kinetic, metal binding, and GDP binding studies with Mg2+ yielded dissociation constants similar to those found with Mn2+. Hence, GDPMH requires one divalent cation per active site to promote catalysis by facilitating the departure of the GDP leaving group, unlike its homologues the MutT pyrophosphohydrolase, which requires two, or Ap4A pyrophosphatase, which requires three.     

Kinetic and magnetic resonance studies of the glutamate-43 to serine mutant of staphylococcal nuclease

The Glu-43 residue of staphylococcal nuclease has been proposed to function as a general base that facilitates the attack of water on the phosphodiester substrate [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555]. With DNA as substrate, Vmax in the glutamate-43--serine (E43S) mutant enzyme is decreased by 2700-fold at pH 7.4 but only 376-fold at pH 9.9. With the wild-type enzyme, Vmax increases with pH to pH 9.2, above which it becomes less sensitive to further increase in pH, leveling off at pH 9.8. In contrast, Vmax of the E43S mutant continues to rise, first order in [OH-], to pH 9.8. Above pH 10 both activities fall irreversible. Hence the hydroxyl ion can partially replace the effect of Glu-43 on kcat, in accord with the proposed role of Glu-43 as a general base. The inflection point in the curve relating pH to log Vmax of the wild-type enzyme at pH 9.4 may reflect the ionization of a Ca2+-bound water, or of a Lys or Tyr residue at the active site. The activator Ca2+ and the competitive inhibitor Mn2+ bind to the E43S mutant an order of magnitude more weakly than to the wild-type enzyme as detected by kinetics and by direct metal binding studies, and approximately one additional water ligand on Mn2+ is found in the binary Mn2+ complex of the E43S mutant (1.4 +/- 0.2) as compared to that of the wild-type enzyme (0.8 +/- 0.2). These data suggest that Glu-43 coordinates the divalent cation in the binary enzyme-metal complex but dissociates from the metal to create a water binding site and to function as a general base in the ternary enzyme-metal-DNA complex. While a 2-fold weaker binding of DNA to the Ca2+ complex of the E43S mutant than to the wild-type enzyme is found by kinetic studies, an order of magnitude tighter binding of the competitive inhibitor 3',5'-pdTp to the Mn2+ and Ca2+ complexes of E43S is found by direct binding studies. Distances from Co2+ to phosphorus in the ternary enzyme-Co2+-pdTp complexes reveal coordination of only the 5'-phosphate by Co2+ on the wild-type enzyme but coordination of both the 3'- and 5'-phosphates of pdTp on the E43S mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of the isolated high-affinity (Ca2+ + Mg2+) ATPase of the human erythrocyte membrane

Solubilized and purified high-affinity (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the human erythrocyte membrane (Wolf, H.U., Dieckvoss, G. and Lichtner, R. (1977) Acta Biol. Ger. 36, 847) has been phosphorylated and dephosphorylated under various conditions with respect to Ca2+ and Mg2+ concentrations. In the range, 0.001--100 mM, the rate of phosphorylation was dependent on Ca2+ concentration, showing a maximum at 10 mM. The phosphorylation rate was nearly independent of the Mg2+ concentration within the range 0.01-1 mM. This enzyme has at least three Ca2+ binding sites with different affinities and regulatory functions: (1) binding to the high-affinity site yields phosphorylation of the enzyme; (2) binding to a low-affinity site (Ca2+ concentrations higher than 40 microM) inhibits dephosphorylation or the conformational change which is necessary for dephosphorylation; (3) by binding to an additional low-affinity site, Ca2+ at concentrations higher than 1 mM abolishes negative cooperative behaviour (shown below 1 mM Ca2+) and causes weak positive cooperativity between at least two catalytic subunits in the phosphorylation reaction. The phosphoprotein obtained at Ca2+ concentrations above 1 mM dephosphorylates spontaneously after removal of the divalent metal ions. Addition of Mg2+ accelerates the dephosphorylation rate. Affinities of the inhibitory Ca2+ binding sites are reduced by the binding of substrate or K+.     

BOOK REVIEW…

Books Review in this article:
Dawes, Ben, ed. 1977. Advances in Parasitology. Vol. 15. Academic Press, 111 Fifth Ave., New York NY 10003, USA. xx + 409 pp. $36.25.
Kcan, H . H., Mott, I(. E. S: Russell. A. J. eds. 1978. Tropical Medicine and Parasitology. Classic Ii1ccstigation.c. 1'01s. I and 11. Corncll Univ. Press, 124 Roberts PI., Ithaca XY 14850. xxiii + G77 pp. $.50.00.
Reissig, J. L., ed. 1977. Microbial Interactions. Vol. 3 of Receptors and Recognition, Series B. Chapman and Hall, London. + 436 pp. $39.50.
'Taylor, Angela E. R. Sr Muller. R.: eds. 1978. Thc Rclccance of ParaJitology to Human Welfare Today. Symbosiuin .Vo. 16 of the British Society for Pnrasitolo, qy. Blackwell Scientific Puhlications, Osney Mead, Oxford 0 5 2 OEL. England and J. B. Lippincott Co., East Washington Squaw, Philadelphia PA 191 0.5, L viii + 135 pp. $18.75.     

Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme.

The solution structure of an RNA hairpin modelling the P5 helix of a group I intron, complexed with Co(NH3)63+, has been determined by nuclear magnetic resonance. Co(NH3)63+, which possesses a geometry very close to Mg(H2O)62+, was used to identify and characterize a Mg2+binding site in the RNA. Strong and positive intermolecular nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH3)63+molecule is in the major groove of tandem G.U base-pairs. The structure of the RNA is characterized by a very low twist angle between the two G.U base-pairs, providing a flat and narrowed major groove. The Co(NH3)63+, although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Negative and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn2+titrations of P5 show specific broadening of protons of the G.U base-pairs that form the metal ion binding site, in agreement with the NOE data from Co(NH3)63+. Binding constants for the interaction of Co(NH3)63+and of Mg2+to P5 were determined by monitoring imino proton chemical shifts during titration of the RNA with the metal ions. Dissociation constants are on the order of 0.1 mM for Co(NH3)63+and 1 mM for Mg2+. Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G.U base-pairs. The affinities of Co(NH3)63+and Mg2+for the tandem G.U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G.C or A.U for G.U pairs also affected the binding, as expected. These structural and thermodynamic results provide systematic new information about major groove metal ion binding in RNA.     

Substitution studies of the second divalent metal cation requirement of protein tyrosine kinase CSK

In addition to a magnesium ion needed to form the ATP-Mg complex, we have previously determined that at least one more free Mg2+ ion is essential for the activation of the protein tyrosine kinase, Csk [Sun, G., and Budde, R. J. A. (1997) Biochemistry 36, 2139-2146]. In this paper, we report that several divalent metal cations, such as Mn2+, Co2+, Ni2+, and Zn2+ bind to the second Mg2+-binding site of Csk with up to 13200-fold higher affinity than Mg2+. This finding enabled us to substitute the free Mg2+ at this site with Mn2+, Co2+, Ni2+, or Zn2+ while keeping ATP saturated with Mg2+ to study the role of the free metal cation in Csk catalysis. Substitution by these divalent metal cations resulted in varied levels of Csk activity, with Mn2+ even more effective than Mg2+. Co2+ and Ni2+ supports reduced levels of Csk activity compared to Mg2+. Zn2+ has the highest affinity for the second Mg2+-binding site of Csk at 0.65 microM, but supports no kinase activity, acting as a dead-end inhibitor. The inhibition by Zn2+ is reversible and competitive against free Mg2+, noncompetitive against ATP-Mg, and mixed against the phosphate accepting substrate, polyE4Y, significantly increasing the affinity for this substrate. Substitution of the free Mg2+ with Mn2+, Co2+, or Ni2+ also results in lower Km values for the peptide substrate. These results suggest that the divalent metal activator is an important element in determining the affinity between Csk and the phosphate-accepting substrate.     

Photocleavage of myosin subfragment 1 by vanadate

The heavy chain of myosin's subfragment 1 (S1) was cleaved at two distinct sites (termed V1 and V2) after irradiation with UV light in the presence of millimolar concentrations of vanadate and in the absence of nucleotides or divalent metals. The V1 site cleavage appeared to be identical with the previously described active site cleavage at serine-180, which is effected by irradiation of a photomodified form of the S1-MgADP-Vi complex [Cremo, C. R., Grammer, J. C., & Yount, R. G. (1989) J. Biol. Chem. 264, 6608-6011]. The V2 site was cleaved specifically, without cleavage at the V1 site, first by formation of the light-stable S1-Co2+ADP-Vi complex at the active site [Grammer, J. C., Cremo, C. R., & Yount, R. G. (1988) Biochemistry 27, 8408-8415] and then by irradiation in the presence of millimolar vanadate. By gel electrophoresis, the V2 site was localized to a region about 20 kDa from the COOH terminus of the S1 heavy chain. From the results of tryptic digestion experiments, the COOH-terminal V2 cleavage peptide appeared to contain lysine-636 in the linker region between the 50- and 20-kDa tryptic peptides of the heavy chain. This site appeared to be the same site cleaved by irradiation of S1 (not complexed with Co2+ADP-Vi) in the presence of millimolar vanadate as previously described [Mocz, G. (1989) Eur. J. Biochem. 179, 373-378]. Cleavage at the V2 site was inhibited by Co2+ but was not significantly affected by the presence of nucleotides or Mg2+ ions. Tris buffer significantly inhibited V2 cleavage. From the results of UV-visible absorption, 51V NMR, and frozen-solution EPR spectral experiments, it was concluded that irradiation with UV light reduced vanadate +5 to the +4 oxidation state, which was then protected from rapid reoxidation by O2 by complexation with the Tris buffer. The relatively stable reduced form or forms of vanadium were not competent to cleave S1 at either the V1 or the V2 site. 51V NMR titration experiments indicated that a tetrameric species of vanadium preferentially bound to S1 and to the S1-MgADP-Vi complex, whereas no binding of either the monomeric or dimeric species could be detected. These results suggest that the vanadate tetramer was responsible for the photocleavage of S1 which occurred at both the V1 and V2 sites in the absence of nucleotides or divalent metals.     

Cobalt hexammine inhibition of the hammerhead ribozyme

The effects of Co(NH(3))(6)(3+) on the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal denaturation studies. Co(NH(3))(6)(3+) efficiently displaces Mn(2+) bound to the ribozyme with an apparent dissociation constant of K(d app) = 22 +/- 4.2 microM in 500 microM Mn(2+) (0.1 M NaCl). Displacement of Mn(2+) coincides with Co(NH(3))(6)(3+) inhibition of hammerhead activity in 500 microM Mn(2+), reducing the activity of the WT hammerhead by approximately 15-fold with an inhibition constant of K(i) = 30.9 +/- 2.3 microM. A residual 'slow' activity is observed in the presence of Co(NH(3))(6)(3+) and low concentrations of Mn(2+). Under these conditions, a single Mn(2+) ion remains bound and has a low-temperature EPR spectrum identical to that observed previously for the highest affinity Mn(2+) site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R. , Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturation experiments also reveal structural effects that accompany the observed inhibition of cleavage and Mn(2+) displacement induced by addition of Co(NH(3))(6)(3+). Taken together, the data indicate that a high-affinity Co(NH(3))(6)(3+) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a model in which at least two types of metal sites, one of which requires inner-sphere coordination, support hammerhead activity.     

The complete amino-acid sequence of a trout-testis non-histone protein, H6, localized in a subset of nucleosomes and its similarity to calf-thymus non-histone proteins HMG-14 and HMG-17.

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].     

Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840-1849 of 18S rRNA, but in the complexes formed with participation of Phe-TPHKPhe (where the G residue carrying the arylazido group occupied position-3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position-3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.     

Site of action of RNase I on the 50 S ribosome of Escherichia coli and the association of the enzyme with the partially degraded subunit.

The 50 S ribosome of Escherichia coli is partially degraded by RNase I in presence of a high concentration of Mg2+ (10 to 20 mM); the partially degraded subunit becomes resistant to the further action of RNase I. The latter remains latent in association with the subparticle as in case of 30 S ribosome (Neu, H.C., and Heppel, L.A. (1954) Proc. Natl. Acad. Sci. U.S.A. 51, 1267-1274). As a result of nucleolytic action, 23 S RNA is degraded to a smaller size and four proteins (L4, L10, L7/L12) are released from the subunit. From the location of these proteins, it appears that the primary site of action of RNase I is the central protuberance of the armchair model proposed for the subunit (Stoffler, G., and Whitman, H.G. (1977) in Molecular Mechanisms of Protein Biosynthesis (Weissbach, H., and Pestka, S., eds) pp. 117-144, Academic Press, New York).     

In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.     

Mitogen-activated 70K S6 kinase. Identification of in vitro 40 S ribosomal S6 phosphorylation sites

Recently we purified and cloned the mitogen/oncogene-activated Mr 70,000 (70K) S6 kinase from the livers of rats treated with cycloheximide (Kozma, S. C., Lane, H. A., Ferrari, S., Luther, H., Siegmann, M., and Thomas, G. (1989) EMBO J. 8, 4125-4132; Kozma, S. C., Ferrari, S., Bassand, P., Siegmann, M., Totty, N., and Thomas, G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7365-7369). Prior to determining the ability of this kinase to phosphorylate the same sites observed in S6 in vivo, we established the effects of different cations and autophosphorylation on kinase activity. The results show that the 70K S6 kinase is dependent on Mg2+ for activity and that this requirement cannot be substituted for by Mn2+. Furthermore, 50-fold lower concentrations of Mn2+ block the effect of Mg2+ on the kinase. This effect is not limited to Mn2+ but can be substituted for by a number of cations, with Zn2+ being the most potent inhibitor, IC50 approximately 2 microM. In the presence of optimum Mg2+ concentrations the enzyme incorporates an average of 1.2 mol of phosphate/mol of kinase and an average of 3.7 mol of phosphate/mol of S6. The autophosphorylation reaction appears to be intramolecular and leads to a 25% reduction in kinase activity toward S6. In the case of S6 all of the sites of phosphorylation are found to reside in a 19-amino acid peptide at the carboxyl end of the protein. Four of these sites have been identified as Ser235, Ser236, Ser240, and Ser244, equivalent to four of the five sites previously observed in vivo (Krieg, J., Hofsteenge, J., and Thomas, G. (1988) J. Biol. Chem. 263, 11473-11477). A fifth mole of phosphate is incorporated at low stoichiometry into the peptide, but the amino acid which is phosphorylated cannot be unequivocally assigned. The low level of phosphorylation of the fifth site in vitro is discussed with regard to known results and to a potential three-dimensional model for the carboxyl terminus of S6.