Biomedical subjects

A S Mildvan

Publications and source records attributed to A S Mildvan.

At least 73 records · Page 4Linked to original sources

PubMed1992

NMR docking of a substrate into the X-ray structure of staphylococcal nuclease.

The conformation of the staphylococcal nuclease-bound metal-dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425-7437] was docked into the X-ray structure of the enzyme-Ca(2+)-3',5'-pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183-201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme-La(3+)-dTdA complex were sequence specifically assigned by 2D phase-sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronuclear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY-HMQC spectroscopy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme-La(3+)-3',5'-pdTp complex and the enzyme-Ca(2+)-3',5'-pdTp complex, proton resonances of Tyr-85, Tyr-113, Tyr-115, and Phe-34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr-113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme-bound dTdA shows the 5'-dA leaving group to partially overlap the inhibitor, 3',5'-pdTp (in the X-ray structure). The 3'-TMP moiety of dTdA points toward the solvent in a channel defined by Ile-18, Asp-19, Thr-22, Lys-45, and His-46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu-43 and to attack the phosphorus with inversion. Arg-35 and Arg-87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg-87 positioned to protonate the leaving 5'-oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5'-dGMP onto the 3'-oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu-80, Asp-83, Lys-84, and Tyr-115 with its 3'-OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide.

Binding Sites↗

PubMed1991

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.

Binding Sites↗

PubMed1991

Studies of the catalytic mechanism of an active-site mutant (Y14F) of delta 5-3-ketosteroid isomerase by kinetic deuterium isotope effects.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the conversion of androst-5-ene-3,17-dione to androst-4-ene-3,17-dione by a stereoselective transfer of the 4 beta-proton to the 6 beta-position. The rate-limiting step has been shown to be the concerted enolization of the enzyme-bound substrate comprising protonation of the 3-carbonyl oxygen by Tyr-14 and abstraction of the 4 beta-proton by Asp-38 [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500]. Primary, secondary, solvent, and combined kinetic deuterium isotope effects have been used to investigate the mechanism of the Y14F mutant, which lacks the proton donor and is 10(4.7)-fold less active catalytically than the wild-type enzyme. With [4 beta-D]androst-5-ene-3,17-dione as a substrate in H2O, a lag in product formation is observed which approaches, by a first-order process, the rate observed with protonated substrate. With the protonated substrate in D2O, a burst in product formation is detected by derivative analysis of the kinetic data which approaches the rate observed with the 4 beta-deuterated substrate in D2O. The absence of such lags or bursts with the protonated substrate in H2O or with the 4 beta-deuterated substrate in D2O, as well as the detection of buffer catalysis by phosphate at pH 6.8, indicates that one or more intermediates dissociate from the enzyme and partition to substrate 31.6 times faster than to product.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Conformation of an enzyme-bound substrate of staphylococcal nuclease as determined by NMR.

The dinucleoside phosphodiester dTdA is a slow substrate of staphylococcal nuclease (kcat = 3.8 X 10(-3) s-1) that forms binary E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. The enzyme enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of six proton resonances of dTdA, and these effects are abolished by binding of the competitive inhibitor 3',5'-pdTp. From paramagnetic effects of Co2+ on 1/T2 of phosphorus, koff of dTdA from the ternary E-Co(2+)-dTdA complex is greater than or equal to 4.8 X 10(4) s-1 and kon greater than or equal to 1.4 X 10(6) M-1 s-1, indicating the 1/T1 values to be in fast exchange. From paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with use of a correlation time of 1.6 ps on the basis of 1/T1 values at 250 and 600 MHz, 7 metal-nucleus distances and 9 lower-limit metal-nucleus distances are calculated. The long Co2+ to 31P distance of 4.1 +/- 0.9 A, which is intermediate between that expected for direct phosphoryl coordination (3.31 +/- 0.02 A) and a second sphere complex with an intervening water ligand (4.75 +/- 0.02 A), suggests either a distorted inner sphere complex or the rapid averaging of 18% inner sphere and 82% second sphere complexes and may explain the reduced catalytic activity with small dinucleotide substrates. Seventeen interproton distances and 108 lower limit interproton distances in dTdA in the ternary E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. While metal-substrate and interproton distances alone did not yield a unique structure, the combination of both sets of distances yielded a very narrow range of conformations for enzyme-bound dTdA, which was highly extended, with no base stacking, with high-anti glycosidic torsional angles for dT (64 degrees less than or equal to chi less than or equal to 73 degrees) and dA (66 degrees less than or equal to chi less than or equal to 68 degrees) and predominantly C-2'-endo sugar puckers for both nucleosides. Although the individual nucleosides are like those of B-DNA, their unstacked conformation, which is inappropriate for base pairing, as well as the conformational angles alpha and gamma of dA and zeta of dT, rule out B-DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗

PubMed1991

Interactions of the acid and base catalysts on staphylococcal nuclease as studied in a double mutant.

The mechanism of the phosphodiesterase reaction catalyzed by staphylococcal nuclease is believed to involve concerted general acid-base catalysis by Arg-87 and Glu-43. The mutual interactions of Arg-87 and Glu-43 were investigated by comparing kinetic and thermodynamic properties of the single mutant enzymes E43S (Glu-43 to Ser) and R87G (Arg-87 to Gly) with those of the double mutant, E43S + R87G, in which both the basic and acidic functions have been inactivated. Denaturation studies with guanidinium chloride, CD, and 600-MHz 1D and 2D proton NMR spectra, indicate all enzyme forms to be predominantly folded in absence of the denaturant and reveal small antagonistic effects of the E43S and R87G mutations on the stability and structure of the wild-type enzyme. The free energies of binding of the divalent cation activator Ca2+, the inhibitor Mn2+, and the substrate analogue 3',5'-pdTp show simple additive effects of the two mutations in the double mutant, indicating that Arg-87 and Glu-43 act independently to facilitate the binding of divalent cations and of 3',5'-pdTP by the wild-type enzyme. The free energies of binding of the substrate, 5'-pdTdA, both in binary E-S and in active ternary E-Ca(2+)-S complexes, show synergistic effects of the two mutations, suggesting that Arg-87 and Glu-43 interact anticooperatively in binding the substrate, possibly straining the substrate by 1.6 kcal/mol in the wild-type enzyme. The large free energy barriers to Vmax introduced by the R87G mutation (delta G1 = 6.5 kcal/mol) and by the E43S mutation (delta G2 = 5.0 kcal/mol) are partially additive in the double mutant (delta G1+2 = 8.1 kcal/mol). These partially additive effects on Vmax are most simply explained by a cooperative component to transition state binding by Arg-87 and Glu-43 of -3.4 kcal/mol. The combination of anticooperative, cooperative, and noncooperative effects of Arg-87 and Glu-43 together lower the kinetic barrier to catalysis by 8.1 kcal/mol.

Arginine↗

PubMed1991

Catalytic mechanism of an active-site mutant (D38N) of delta 5-3-ketosteroid isomerase. Direct spectroscopic evidence for dienol intermediates.

The delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the conversion of androst-5-ene-3,17-dione to androst-4-ene-3,17-dione by a stereospecific transfer of the 4 beta-proton to the 6 beta-position. The reaction involves two steps: (a) a rate-limiting concerted enolization, comprising protonation of the 3-carbonyl oxygen by the phenolic hydroxyl group of Tyr-14 and abstraction of the 4 beta-proton by the carboxylate group of Asp-38, and (b) rapid reketonization of the dienol, which may or may not be concerted. The active-site mutant D38N, which lacks the base responsible for proton transfer, is about 10(6.0)-fold less active catalytically than the wild-type enzyme. With the D38N mutant it was demonstrated spectroscopically that the enzymatic reaction involves the conversion of the substrate to both the dienol and its anion as tightly enzyme-bound intermediates, which are then converted much more slowly to the alpha,beta-unsaturated product. In contrast to the mechanism of the wild-type enzyme, the enolization reaction promoted by the D38N mutant is not stereospecific with respect to removal of the 4 beta-proton and shows primary kinetic isotope effects on enolization when either 4 alpha or 4 beta or both of these protons are replaced by deuterium. Kinetic isotope effects obtained with deuterated substrates, solvent, or combinations of the two indicate that, unlike in the wild-type enzyme, protonation of the carbonyl oxygen and removal of the C-4 proton are not concerted in the D38N mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗

PubMed1991

Stereochemistry of the concerted enolization catalyzed by delta 5-3-ketosteroid isomerase.

The reaction catalyzed by delta 5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the delta 5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A.S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A.S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in kcat (12,000 s-1) and a 3.6-fold decrease in KM (94 microM) for delta 5-androstene-3, 17,dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pKa of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pKa values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5-and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2 alpha-, 2 beta-, or 6 beta-proton resonances of the bound steroid but no NOE's to the 4- or 6 alpha-protons of the steroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Escherichia coli↗

PubMed1990

Combined effects of two mutations of catalytic residues on the ketosteroid isomerase reaction.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position with Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149-159]. Primary, secondary, and combined deuterium kinetic isotope effects establish concerted substrate enolization to be the rate-limiting step with the wild-type enzyme [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500]. The product of the fractional kcat values resulting from the Y14F mutation (10(-4.7)) and the D38N mutation (10(-5.6)) is comparable (10(-10.3)) to that of the double mutant Y14F + D38N (less than or equal to 10(-10.4)) which is completely inactive. Hence, the combined effects are either additive or synergistic. Quantitatively, similar effects of the two mutations on kcat/KM are found in the double mutant. Despite its inactivity, the Y14F + D38N double mutant forms crystals indistinguishable in form from those of the wild-type enzyme, tightly binds steroid substrates and substrate analogues, and immobilizes a spin-labeled steroid in an orientation indistinguishable from that found in the wild-type enzyme, indicating that the double mutant is otherwise largely intact. It is concluded that the total enzymatic activity of ketosteroid isomerase probably results from the independent and concerted functioning of Tyr-14 and Asp-38 in the rate-limiting enolization step, in accord with the perpendicular or antarafacial orientation of these two residues with respect to the enzyme-bound substrate. Synergistic effects of mutating two residues on kcat and on kcat/KM of enzyme-catalyzed multistep reactions are shown, theoretically, to occur when both residues act independently in the same step, and simple additivity occurs when this step is rate-limiting. Other conditions for additivity of the effects of mutations of kcat and kcat/KM are theoretically explored.

Binding Sites↗

PubMed1990

Diverse interactions between the individual mutations in a double mutant at the active site of staphylococcal nuclease.

In principle, the quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on the kinetic or thermodynamic parameter measured, the D21E and R87G mutations of staphylococcal nuclease exhibit four of these five categories of interaction in the double mutant. While Vmax of the R87G single mutant of staphylococcal nuclease is 10(4.8)-fold lower than that of the wild-type enzyme and the Vmax of the D21E single mutant is 10(3.0)-fold below that of wild type, the double mutant D21E + R87G was found to lose a factor of only 10(4.1) in Vmax relative to wild type, rather than the product of the two single mutations (10(7.8)). These results suggest antagonistic structural effects of the individual R87G and D21E mutations. An alternative explanation for the nonadditivity of effects, namely, the separate functioning of these residues in a stepwise mechanism involving the prior attack of water on phosphorus followed by protonation of the leaving group by Arg-87, is unlikely since no enzyme-bound phosphorane intermediate (less than 1% of [enzyme]) was found under steady-state conditions on the R87G mutant by 31P NMR at 242.9 MHz. Like the effects on Vmax, quantitatively similar antagonistic effects of the two mutations were detected on the binding of divalent cations in binary enzyme-Ca2+ and enzyme-Mn2+ complexes and in the ternary enzyme-Ca2(+)-5'-pdTdA complex, suggesting that the effects on Vmax result from antagonistic structural changes at the Ca2+ binding site. Simple additive weakening effects of the two mutations were found on the binding of the substrate 5'-pdTdA, in both the absence and the presence of the divalent cations, Mn2+ and Ca2+. However, synergistic effects of the two mutations were found on the binding of the substrate analogue 3',5'-pdTp, profoundly weakening its binding to the double mutant in both the absence and the presence of divalent cations. Such synergistic effects of the two mutations may result from negative cooperativity or strain in the binding of 3',5'-pdTp to the wild-type enzyme. It is concluded that the quantitative interactions of two active-site mutations of an enzyme can vary greatly depending on which parameter of the enzyme is measured. When the two mutations interact in the same way on several parameters, a common underlying mechanism is suggested.

Binding Sites↗

PubMed1990

Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment.

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1990

Studies of the mechanism of the delta 5-3-ketosteroid isomerase reaction by substrate, solvent, and combined kinetic deuterium isotope effects on wild-type and mutant enzymes.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position using Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149]. On deuteration of the 4 beta-position (97.0%) of the substrate, kcat(H)/kcat(4 beta-D) is 6.1 in H2O and 6.3 in D2O. The solvent isotope effect, kcat(H2O)/kcat(D2O), is 1.6 for both the 4 beta-H and 4 beta-D substrates. Mutation of Tyr-55 to Phe lowers kcat 4.3-fold; kcat(H)/kcat/4 beta-D) is 5.3 in H2O and 5.9 in D2O, and kcat(H2O)/kcat(D2O) with the 4 beta-H and 4 beta-D substrates is 1.5 and 1.7, respectively, indicating concerted general acid-base catalysis in either the enolization or the ketonization step of both the wild-type and the Tyr-55----Phe (Y55F) mutant enzymes. An additional slow step occurs with the Y55F mutant. Smaller isotope effects on Km are used to estimate individual rate constants in the kinetic schemes of both enzymes. On deuteration of the 4 alpha-position (88.6%) of the substrate, the secondary isotope effect on kcat/Km corrected for composition is 1.11 +/- 0.02 with the wild-type enzyme and 1.12 +/- 0.02 with the Y55F mutant. These effects decrease to 1.06 +/- 0.01 and 1.07 +/- 0.01, respectively, when the 4 beta-position is also deuterated, thereby establishing these to be kinetic (rather than equilibrium) secondary isotope effects and to involve a proton-tunneling contribution. Deuteration of the 6-position of the substrate (92.0%) produces no kinetic isotope effects on kcat/Km with either the wild-type (1.00 +/- 0.01) or the Y55F mutant (1.01 +/- 0.01) enzyme. Since a change in hybridization from sp3 to sp2 occurs at C-4 only during enolization of the substrate and a change in hybridization at C-6 from sp2 to sp3 occurs only during reketonization of the dienol intermediate, enolization of the substrate constitutes the concerted rate-limiting step. Concerted enolization is consistent with the right angle or antarafacial orientations of Tyr-14 and Asp-38 with respect to the enzyme-bound substrate and with the additive effects on kcat of mutation of these catalytic residues [Kuliopulos, A., Talalay, P., & Mildvan, A. S. (1990) Biophys. J. 57, 39a].

Deuterium↗

PubMed1990

NMR studies of the active site of DNA polymerase I and of a 50-residue peptide fragment of the enzyme.

Transferred nuclear Overhauser effects (NOEs) and selective T1 measurements were used to determine interproton distances in the substrates Mg2+dATP and Mg2+TTP bound to the large fragment of DNA polymerase I (Pol I). The distances are consistent with high anti, O1' endo conformations for the enzyme-bound substrates, similar to nucleotides of B-DNA. These substrate conformations show little or no change when the complementary RNA templates (rU)57 or (rA)50 are bound. In contrast, multiple conformations, including syn and anti species, are required to fit the interproton distances measured on the enzyme-bound guanine nucleotide substrates Mg2+dGTP and Mg2+ddGTP. These multiple substrate conformations simplify to a single high anti, O1' endo conformation when the complementary template (rC)37 is bound, possibly due to base-pairing with the template, as in the active complex. In the presence of both template and primer, enzyme-bound Mg2+ddGTP reverts to multiple conformations. This ability of Pol I to decrease the fraction of bound substrate which is appropriate for primer elongation may be an error-preventing mechanism. In all cases, the conformations of the average nucleotide of the enzyme-bound RNA templates are also B-like. Transferred NOEs from protons of the enzyme to those of bound dNTP substrates suggest hydrophobic (Ile, Leu) and an aromatic amino acid (Tyr) at the substrate binding site. Peptide I, a synthetic 50-residue peptide based on residues 728 to 777 of the Pol I sequence, containing the conserved sequence L-I-Y-G, retains significant secondary and tertiary structure in solution as found by circular dichroism (CD) and 2D NMR. While the X-ray structure shows 48% helix in this region, the sequence specific NOESY analysis suggests 18% helix, and the preservation of two of the three beta turns. Peptide I shows tight binding of dNTP substrates, the substrate analog 2',3'-trinitrophenyl-ATP, and duplex DNA, providing direct evidence that the active site for polymerization lies in this region of the enzyme, with the substrate binding along the O-helix near Leu-764, Ile-765, and Tyr-766. Another synthetic peptide, peptide II, based on residues 840 to 888 of the Pol I sequence also retains much secondary structure as detected by CD but does not bind the substrate analog TNP-ATP.

Adenosine Triphosphate↗

PubMed1990

Involvement of a divalent cation in the binding of fructose 6-phosphate to Trypanosoma cruzi phosphofructokinase: kinetic and magnetic resonance studies.

When Mg2+ ions were replaced by Mn2+ in the assay of Trypanosoma (Schizotrypanum) cruzi phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) the Km for D-fructose 6-phosphate (F6P) was reduced threefold while the corresponding constant for ATP was essentially unaffected. A detailed kinetic investigation showed that the apparent Km for F6P decreased monotonically with increasing free Mn2+ concentrations, from a limiting value of 5.7 mM in its absence to a limiting value of 1.1 mM in the presence of saturating concentrations of the ion; the Vmax of the enzyme was, on the other hand, not affected by the concentration of Mn2+. Conversely, it was shown that the apparent Km for Mn2+ at fixed MnATP concentrations decreased with increasing F6P concentrations, from a limiting value of 30 microM in the absence of the sugar phosphate to 9 microM at saturating concentrations of the substrate, while the apparent Vmax increased monotonically from zero to its limiting value. Both electron paramagnetic resonance and water proton longitudinal relaxation studies showed binding of one Mn2+ ion per 18,000 Da catalytic subunit of enzyme in the absence of F6P, with a dissociation constant of 57 +/- 4 microM, comparable to the apparent Km for the ion in the absence of F6P. The presence of saturating level of F6P decreases the value of the dissociation constant of Mn2+ to a limiting value of 7.9 microM in agreement with the results of the kinetic analysis. The substrate F6P decreases the enhancement of the water proton longitudinal relaxation rate in a saturable fashion, suggesting displacement of water molecules coordinated to the enzyme-bound Mn2+ ion by the sugar phosphate. Computer fitting of the several dissociation constants and relaxation enhancements for binary and ternary complexes gives a value of 7.9 mM for the dissociation constant of the enzyme-F6P complex in the absence of Mn2+ and 1.1 mM in the presence of saturating concentrations of the ion, in excellent agreement with the respective Km values of F6P extrapolated to zero and saturating Mn2+, respectively. Studies of the frequency dependence of the water proton longitudinal relaxation rate enhancements in the presence of both binary (enzyme-Mn2+) and ternary (enzyme-Mn2(+)-F6P) complexes, are most simply explained by assuming two exchangeable water molecules in the coordination sphere of the enzyme-bound Mn2+ in the binary complex, while in the ternary complex the data are consistent with the displacement of one of the water molecule from the coordination sphere with no significant alteration of the correlation time. Overall, the kinetic and binding data are consistent with the formation of an enzyme-metal-F6P bridge complex at the active site of T. cruzi phosphofructokinase, a coordination scheme which is unique among the phosphofructokinases.

Animals↗

PubMed1989

Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme.

The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme.

Adenosine Triphosphate↗

PubMed1989

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)

Cations, Divalent↗

PubMed1989

Kinetic and ultraviolet spectroscopic studies of active-site mutants of delta 5-3-ketosteroid isomerase.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes the highly efficient isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-A X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E.M., Talalay, P., & Mildvan, A.S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E.M., Piro, O.E., & Sigler, P.B. (1984) J. Biol. Chem. 259, 9096-9103]. The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 10(5.6)-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4 beta-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the Y14F mutant shows a 10(4.7)-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S.F., Kawahara, F.S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1989

Recognition in cell adhesion. A comparative study of the conformations of RGD-containing peptides by Monte Carlo and NMR methods.

Many of the proteins that mediate cell adhesion processes processes-fibronectin, fibrinogen, vitronectin, von Willebrand factor, osteopontin, laminin and various collagens--contain the amino acid sequence Arg-Gly-Asp. Short peptides that include this sequence have been shown to inhibit the interactions of cell adhesion proteins with their receptors and to have dramatic effects on developmental processes involving cellular recognition. In order to determine which conformations are accessible to Arg-Gly-Asp containing peptides, we analyzed tri-, tetra- and pentapeptides using molecular mechanics and Monte Carlo methods, and studied the solution conformations of the pentapeptide Gly-Arg-Gly-Asp-Ser using nuclear magnetic resonance techniques. The Monte Carlo method was used to: (a) identify the low energy conformations of the peptides and (b) evaluate their thermodynamic properties. In the case of the pentapeptide Gly-Arg-Gly-Asp-Gly, the four stable conformations include three with reverse turns and one open structure. The conformations found in this analysis are compatible with the nuclear magnetic resonance (nuclear Overhauser effect) data.

Amino Acid Sequence↗

PubMed1989

Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for the native enzyme) (Hamada and Kuby, 1978), and the K'd measured for Cr3+ ATP [corrected] and the synthetic peptide I1-45 (Fry et al., 1985b).

Adenosine Diphosphate↗