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A S Mildvan

Publications and source records attributed to A S Mildvan.

At least 55 records · Page 3Linked to original sources
PubMed1995

Enzymatic and nonenzymatic polarizations of alpha,beta-unsaturated ketosteroids and phenolic steroids. Implications for the roles of hydrogen bonding in the catalytic mechanism of delta 5-3-ketosteroid isomerase.

Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17 beta-estradiol and 17 beta-dihydroequilenin), which are potent competitive inhibitors of delta 5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme-steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17 beta-estradiol and 17 beta-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a lambda max of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a lambda max of 258.5 nm in aqueous solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗
PubMed1994

Vaccinia DNA topoisomerase I: kinetic evidence for general acid-base catalysis and a conformational step.

The pH dependences of the internal equilibrium (Kcl) and rate constants for site-specific DNA strand cleavage (kcl) and resealing (kr) catalyzed by Vaccinia DNA topoisomerase I have been investigated using single-turnover conditions in the pH range 4.6-9.8 at 20 degrees C. The pH dependence of the rate constant for strand cleavage (kcl) shows a bell-shaped profile with apparent pKa values of 6.3 +/- 0.2 and 8.4 +/- 0.2, suggesting base catalysis of the attack of the active site Tyr-274 on the phosphodiester phosphorus, and acid catalysis of the expulsion of the 5'-deoxyribose oxygen. A low pKa (i.e., 6.3) for Tyr-274 in the free enzyme is ruled out by NMR titration from pH 5.1 to 8.8 monitoring the C-zeta chemical shift of [zeta-13C]-tyrosine-enriched topoisomerase. The dependence of the internal equilibrium constant (Kcl) on pH reveals very similar pKa values as kcl (5.8 +/- 0.2 and 8.6 +/- 0.2). However, kr is found to be independent of pH. The differing response of kcl and kr to pH rules out a simple two-state internal cleavage equilibrium and suggests that a conformational change occurs following formation of the covalent complex which retains the correct protonation state for strand religation. A conformation step is further indicated by a 4.6-fold "thio effect" on kcl upon substitution of the nonbridging oxygen atom of the attacked phosphoryl group by sulfur [Stivers, J. T., Shuman, S., & Mildvan, A. S. (1994) Biochemistry 33, 327], and the absence of such an effect on kr, (krphos/krthio = 0.9 +/- 0.2), indicating the rates of cleavage and religation to be limited by covalent chemistry and a conformational step, respectively. The rate constant of this conformational change in the direction of religation agrees with the average rate constant for supercoil release from plasmid substrates, suggesting this conformational change to be a part of the topoisomerization step. Although the general acid and general base catalysts have not yet been identified, the quantitative roles of these and other residues in catalysis are discussed.

Base Sequence↗
PubMed1994

NMR docking of a substrate into the X-ray structure of the Asp-21-->Glu mutant of staphylococcal nuclease.

To understand the structural basis of the 1500-fold decrease in catalytic activity of the D21E mutant of staphylococcal nuclease in which an aspartate ligand of the essential Ca2+ has been enlarged to glutamate, the conformation of the enzyme-bound substrate dTdA has been determined by NMR methods and has been docked into the X-ray structure of the D21E mutant (Libson, A. M., Gittis, A.G., & Lattman, E. E. Biochemistry, preceding paper in this issue) based on distances from the bound metal ion to dTdA and on intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of dTdA, using energy minimization to relieve small overlaps. Like the wild-type enzyme, the D21E mutant forms binary E-M and E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. D21E enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of four proton resonances of dTdA, and these effects are abolished by the binding of the competitive inhibitor 3',5'-pdTp. From the paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with the use of a correlation time of 1.1 ps based on 1/T1 values at 250 and 600 MHz, five metal-nucleus distances and 11 lower limit metal-nucleus distances have been calculated. The Co2+ to 31P distance of 4.1 +/- 0.9 A agrees with that found on the wild-type enzyme (Weber, D. J., Mullen, G. P., & Mildvan, A. S. (1991) Biochemistry 30, 7425-7437) and indicates at least 18% inner sphere phosphate coordination. Fourteen interproton distances and 109 lower limit interproton distances in dTdA in the ternary D21E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. Both the metal-nucleus and interproton distances were necessary to compute a narrow range of conformations for enzyme-bound dTdA. As on the wild-type enzyme, the conformation of dTdA on the D21E mutant is highly extended, with high-anti C-2' endo conformations for the individual nucleosides. However, significant conformational differences are found in the torsional angles chi of dA (delta chi = 49 +/- 3 degrees), in gamma of dT (delta gamma = 108 +/- 30 degrees) and in zeta of dT (delta zeta = 124 +/- 38 degrees).(ABSTRACT TRUNCATED AT 400 WORDS)

Cobalt↗
PubMed1994

Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes.

Kinetic analyses of both the Mn(2+)- and Mg(2+)-activated hydrolysis of dGTP by MutT show the requirement for two divalent cations. Whereas Mn2+ supports a 20-fold lower kcat (0.19 s-1) than Mg2+ (4.0 s-1), the Km of Mn2+.dGTP (6.3 microM) is 45-fold lower than that of Mg2+.dGTP (284 microM). Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a Ki for Mg2+.AMPCPP (42 microM) which is 57-fold lower than the Ki of Mg2+.AMPCPP (2.4 mM). Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ complex is the catalytically active species. The 12 dissociation constants describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluated for both Mn2+ and Mg2+, using EPR and NMR methods. MutT binds a single Mn2+ with a Kd of 130 +/- 40 microM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 microM. The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a Kd of 16 +/- 2 microM in agreement with the kinetically determined KmMn2+ of 26 +/- 10 microM. MutT.Mn2+ binds Mn2+.AMPCPP with Kd of 16 +/- 4 microM, whereas MutT alone binds Mn2+.AMPCPP with a Kd of 135 +/- 30 microM. The 17-fold enhanced paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons found with the binary MutT.Mn2+ complex decreases to 4.7-fold upon binding of AMPCPP and to 8.7-fold upon binding of Mn2+.AMPCPP, further supporting a metal-bridge MutT.M2+.NTP.M2+ complex. By competition with Mn2+ MutT binds Mg2+ at one site with a Kd of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a Kd of 0.9 mM. These values are comparable to the kinetically determined KaMg of 15 +/- 7 mM and KmMg of 1.7 +/- 0.7 mM, respectively. Studies with the racemic, substitution-inert beta, gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the lambda stereoisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal ion requirement.

Adenosine Triphosphate↗
PubMed1994

Vaccinia DNA topoisomerase I: single-turnover and steady-state kinetic analysis of the DNA strand cleavage and ligation reactions.

Vaccinia DNA topoisomerase I catalyzes a reversible, site-specific strand cleavage and resealing reaction with duplex DNA involving a transient 3'-phosphotyrosyl linkage between the DNA and Tyr-274 of the enzyme. Single-turnover and steady-state kinetic measurements, as well as DNA binding studies with DNA duplexes containing the preferred cleavage sequence (5'-CCCTT decreases -3') in 50 mM Tris-HCl, pH 7.5, at 20 degrees C, have permitted the evaluation of the individual rate constants for strand cleavage (kcl) and religation (kr) and for duplex DNA binding and dissociation. The values of kcl = 0.07 s-1 and kr = 0.66 s-1 indicate that the internal cleavage equilibrium (Kcl = kcl/kr = 0.1) favors the uncleaved E.DNA complex. The apparent second-order rate constant kcl/KmDNA + = 8 x 10(5) M-1 s-1 for the single-turnover cleavage reaction is 10(2)-10(3)-fold less than the rate of diffusional encounter and provides an estimate of k(on) (DNA). Single-turnover cleavage experiments using a duplex substrate with a nonbridging racemic phosphorothioate nucleotide substitution at the cleavage site showed biphasic cleavage kinetics with equal amplitudes for each phase, which was fit to a double exponential: kfast = 0.01 s-1 and kslow = 0.0004 s-1. These "thio effects" (= kclphos/kclthiol) of 4.6- and 115-fold indicate that cleavage is at least partially rate-limiting in the single-turnover reaction; the two kinetic phases indicate a strong preference for cleavage of one thio isomer. Multiple-turnover cleavage-religation reactions showed an initial pre-steady-state burst proportional to enzyme, followed by a slower steady-state rate with a value of kcat = 0.006 s-1. The phosphorothioate substrate showed a smaller burst and no significant thio effect on kcat. These results indicate fast chemical steps and largely rate-limiting dissociation of the religated product (KD = 54 nM) in the steady-state, a conclusion confirmed by direct measurement of the rate constant for product dissociation as 0.01 s-1. MgCl2 (5 mM) increases this rate constant by an order of magnitude, thus explaining the divalent cation induced acceleration of DNA supercoil relaxation by this enzyme. No divalent cation binding by the enzyme was detected indicating this effect to result from metal binding to DNA. The rate constants kcl and kr for the nucleophilic attack of Tyr-274 and deoxyribose 5'-OH at phosphorus represent approximately 10(9)- and 10(12)-fold enhancements over the analogous attack of phenolate and alcoholate anions at the phosphorus backbone of DNA in solution at pH 7.5. Consistent with this estimate, the Y274F mutant is at least 10(6)-fold less active in cleavage than the wild-type enzyme.

Base Sequence↗
PubMed1994

Magnetic resonance studies of the binding of oligonucleotide substrates to mutants of staphylococcal nuclease.

By a combination of NMR docking and model building, the substrate binding site on staphylococcal nuclease was found to accommodate a trinucleotide and to consist of three subsites, each interacting with a single nucleotidyl unit of DNA. Binding of the essential Ca2+ activator and substrate cleavage occur between subsites 1 and 2. Hence, catalytically productive binding would span subsites 1 and 2 while nonproductive binding would span subsites 2 and 3. Lys-49 is near subsite 1, and Lys-84 and Tyr-115 interact with substrates at subsite 3 [Weber, D.J., Gittis, A.G., Mullen, G.P., Abeygunawardana, C., Lattman, E.E., Mildvan, A.S. Proteins 13:275-287, 1992]. The proposed locations of these subsites were independently tested by the effects of the K49A, K84A, and Y115A mutations of staphylococcal nuclease on the binding of Mn2+, Ca2+, and the dinucleotide and trinucleotide substrates, 5'-pdTdA, dTdA, and dTdAdG. These three mutants have previously been shown to be fully active and to have CD and 2D NMR spectra very similar to those of the wild-type enzyme (Chuang, W.-J., Weber, D.J., Gittis, A.G., Mildvan, A.S. Proteins 17:36-48, 1993). All three mutant enzymes and their pdTdA and dTdA complexes (but not their dTdAdG complex) bind Mn2+ and Ca2+ more weakly than the wild-type enzyme by factors ranging from 2 to 11. The presence of a terminal phosphate as in 5'-pdTdA raises the affinity of the substrate for staphylococcal nuclease and its three mutants by two orders of magnitude and for the corresponding enzyme-metal complexes by three to four orders of magnitude, suggesting that the terminal phosphate is coordinated by the enzyme-bound divalent cation. Such complexation would result in the nonproductive binding of 5'-pdTdA at subsites 2 and 3. Accordingly, the K84A and Y115A mutations significantly weaken the binding of 5'-pdTdA and its metal to staphylococcal nuclease by factors of 2.2 to 37.8, while the K49A mutation has much smaller or no effect. Such nonproductive binding explains the low activity of staphylococcal nuclease with small substrates, especially those with a terminal phosphate. Similarly, the K84A and Y115A mutations weaken the binding of dTdA and its metal complexes to the enzyme by factors of 3.4 to 13.1 while the K49A mutation has smaller effects indicating significant nonproductive binding of dTdA. The trinucleotide dTdAdG binds more tightly to wild-type and mutant staphylococcal nuclease and to its metal complexes than does the dinucleotide dTdA by factors of 2.4 to 12.2, reflecting the occupancy of an additional subsite.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗
PubMed1993

Sequence-specific assignments of the backbone 1H, 13C, and 15N resonances of the MutT enzyme by heteronuclear multidimensional NMR.

The MutT protein, a 129-residue enzyme from Escherichia coli which prevents A.T-->C.G mutations, catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleoside monophosphates (NMP) and pyrophosphate [Bhatnagar, S. K., Bullions, L. C., & Bessman, M. J. (1991) J. Biol. Chem. 266, 9050-9054], by a mechanism involving nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. C., Bessman, M. J., & Mildvan, A. S. (1992a) J. Biol. Chem. 267, 16939-16942]. The bacterial MutT gene was inserted into the plasmid pET-11b under control of the T7 promoter and overexpressed in minimal media, permitting labeling of MutT with 13C and/or 15N. The yield after purification of the soluble fraction was approximately 35 mg of homogeneous MutT/L with physical and enzymatic properties indistinguishable from those of the originally isolated enzyme. Essentially complete sequence-specific assignments of the backbone HN, N, C alpha, H alpha, and CO resonances of the free enzyme (1.5 mM) were made at pH 7.4 and 32 degrees C, by heteronuclear double- and triple-resonance experiments using a modified Bruker AM 600 NMR spectrometer. Specifically, 1H[15N]HSQC, 1H[15N]TOCSY-HMQC, and 1H[15N]NOESY-HMQC experiments were done with uniformly 15N-labeled enzyme. A 1H[15N] HSQC experiment was done with selective [alpha-15N]Lys-labeled enzyme. Also HNCA, HN(CO)CA, HNCO, constant time 1H[13C]HSQC, HCACO, and HCA(CO)N experiments were done with uniformly 13C- and 15N-labeled enzyme. Sequence-specific assignments were initiated from HN and 15N chemical shifts of Gly residues and of selectively labeled Lys residues in 1H[15N]HSQC experiments. They were confirmed by C alpha chemical shifts of Ala residues uniquely identified by residual coupling to C beta resonances in constant time 1H[13C]HSQC experiments. The sequence-specific assignments proceeded bidirectionally, terminating at Pro residues and at residues with undetectable NH signals, and the segments were linked to complete the backbone assignments. The backbone assignments reported here have permitted the interpretation of NOEs in the elucidation of the solution secondary structure of MutT, and the C alpha and H alpha chemical shifts have provided an independent approach to identifying secondary structural elements and to define their extent [Weber, D. J., Abeygunawardana, C., Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (following paper in this issue)].

Amino Acid Sequence↗
PubMed1993

Secondary structure of the MutT enzyme as determined by NMR.

The MutT enzyme (129 amino acids) catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleotides (NMP) and pyrophosphate by nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. L., Bessman, M. J., & Mildvan, A. S. (1992) J. Biol. Chem. 267, 16939-16942]. Backbone NMR assignments for the H alpha, 13C alpha, HN, 15N, and carbonyl 13C' resonances, based on heteronuclear methods have been reported for MutT [Abeygunawardana, C., Weber, D. J., Frick, D. N. Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (preceding paper in this issue)]. Here, we report the secondary structure of MutT in solution on the basis of these assignments, NOE data derived from 2D and 3D homonuclear and heteronuclear NMR spectra, and amide NH exchange data. Consistent with near neighbor NOEs, H alpha and C alpha chemical shifts, and amide exchange rates, MutT contains two alpha-helices spanning residues 47-59 (helix 1) and residues 119-128 (helix 2), respectively. The helical content predicted from NMR (17.8 +/- 1.0%) is consistent with that predicted by circular dichroism spectroscopy (20.9 +/- 5.4%). A mixed parallel and antiparallel beta-sheet with five beta-strands (A-E) consists of residues A, 3-13; B, 18-24; C, 70-74; D, 79-87; and E, 102-106.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1993

Conformation and interaction of phenylalanine with the divalent cation at the active site of human recombinant tyrosine hydroxylase as determined by proton NMR.

Recombinant human tyrosine hydroxylase has been purified as a metal-free apoenzyme (apo-hTH1) which tightly binds one Fe2+, Co2+, or Zn2+ per subunit with activation only by Fe2+ and competitive inhibition by the other cations. L-tyrosine and L-phenylalanine are alternative substrates for this enzyme, giving similar Vmax values, although the KM value for phenylalanine is about 8-fold greater than for tyrosine. Apo-hTH1 enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 values of the protons of enzyme-bound phenylalanine both in the presence and in the absence of the oxidized form of the cofactor L-erythro-7,8-dihydrobiopterin (BH2), which was used as an inactive analog of the natural cofactor (6R)-1-erythro-tetrahydrobiopterin (BH4). No effects of hTH1-Zn2+ on 1/T1 or 1/T2 are found. From paramagnetic effects of hTH1-Co2+ on 1/T1 of phenylalanine protons at 250 and 600 MHz, in the presence of BH2, a correlation time (tau c) of 1.8 +/- 0.1 ps was found. Using this tau c value, and assuming that only one proton of the pairs H3,H5, and H2,H6 is experiencing the total paramagnetic effect (asymmetric limiting case), distances from enzyme-bound Co2+ to phenylalanine (+/- 1.2 A) of 6.1 A (H3 or H5), 6.3 A (H2 or H6), 7.0 A (H4), 7.3 A (H alpha), > or = 7.4 A (H beta-pro-S), and > or = 7.6 A (H beta-pro-R) were calculated. The distances to H3 or H5 and to H2 or H6 are slightly increased to 6.8 and 7.0 A, respectively, if each proton of both degenerate pairs equally experiences the paramagnetic effect of Co2+ (symmetric limiting case). These distances place the aromatic ring of phenylalanine in the second coordination sphere of the metal, which would permit an Fe-bound oxy or peroxy species to approach molecular contact with C3/C4, suggesting a direct role of Fe2+ in the hydroxylation reaction. The same correlation time and similar distances were found in the absence of BH2 with H4 of phenylalanine slightly closer to the metal. In the ternary hTH1-Zn(2+).BH2.phenylalanine complex, eight interproton distances in the enzyme-bound phenylalanine were determined by NOESY spectra at 600 MHz at 35-, 50-, and 75-ms mixing times. The conformation of enzyme-bound phenylalanine, consistent with the six Co(2+)-proton distances and the eight interproton distances, is partially extended with torsional angles chi 1 = 97 degrees +/- 3 degrees and chi 2 = -78 degrees +/- 2 degrees.

Apoenzymes↗
PubMed1993

Environments and mechanistic roles of the tyrosine residues of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and conservative transfer of the 4 beta-proton to the 6 beta-position. The 10(9.5)-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively. The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 +/- 0.2 by UV titration. However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5. This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue. Mutation of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43% and 98% of the wild-type enzyme. Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity. These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca. pKa-7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only. The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme. The unusually high pKa value of Tyr-14 of 11.6 +/- 0.2 was used to estimate a local dielectric constant of 18 +/- 2 near this residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Sequential proton NMR resonance assignments, circular dichroism, and structural properties of a 50-residue substrate-binding peptide from DNA polymerase I.

Peptide I, a 50-amino acid synthetic peptide based on residues 728 to 777 of DNA polymerase I, binds dNTP substrates and duplex DNA (G. Mullen, P. Shenbagamurthi, and A.S. Mildvan, J. Biol. Chem. 264, 19637-19647, 1988). The structural properties of peptide I at pH 3.9 have been studied by CD spectroscopy and by 2D proton NMR at 600 MHz. The CD spectra are fit by assuming that peptide I contains 17% helix, 17% beta-structure, and 66% coil. The substrate dATP binds tightly to peptide I under these conditions (KD = 0.5 microM) as determined by fluorescence quenching but induces no change in peptide conformation, as detected by CD spectroscopy. Proton resonances of peptide I have been assigned by double quantum filtered correlated spectroscopy, total correlated spectroscopy, and nuclear Overhauser effect spectroscopy. As found with other peptides, peptide I is best characterized by both extended and partially folded secondary structures which equilibrate rapidly on the NMR time scale. A region from residues 3 through 10 displays nuclear Overhauser effects (NOEs) consistent with the rapid equilibration of a nascent helix with a random extended structure. Alternatively this segment of residues is consistent with a series of three opened-out turns. A nonclassical turn is found between residues 14 and 17 and from residues 44 to 47, the latter closing irregular antiparallel strands from residues 42 to 48. The remainder of the peptide is a coil. A residue-by-residue comparison of the best-fit solution structure of the peptide with that of the corresponding sequence in the X-ray structure of the complete enzyme reveals that 36% of the amino acids are found to be in a conformation similar to that in the enzyme. Such partial and transient folding of the peptide indicates that the major role of the remainder of the protein is to provide structural support for the active site region of the enzyme. As detected by interresidue NOEs and NOEs to water protons, the homologous sequence Leu-37-Ile-38-Tyr-39-Gly-40, together with Phe-15 of the peptide, provides an exposed hydrophobic cluster of residues which may constitute the substrate binding site. An exposed cluster of cationic residues consisting of Arg-27, Arg-28, Lys-31, and possibly Arg-48 may provide the binding site for duplex DNA.

Adenosine Triphosphate↗
PubMed1993

NMR docking of the competitive inhibitor thymidine 3',5'-diphosphate into the X-ray structure of staphylococcal nuclease.

In the X-ray structure of the ternary staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the bound inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of the enzyme in the crystal lattice (Loll, P. J. and Lattman, E. E. Proteins 5:183-201, 1989; Serpersu, E. H., Hibler, D. W., Gerlt, J. A., and Mildvan, A. S. Biochemistry 28:1539-1548, 1989). Since this interaction does not occur in solution, the NMR docking procedure has been used to correct this problem. Based on 8 Co(2+)-nucleus distances measured by paramagnetic effects on T1, and 9 measured and 45 lower limit interproton distances determined by 1D and 2D NOE studies of the ternary Ca2+ complex, the conformation of enzyme-bound 3',5'-pdTp is high-anti (chi = 58 +/- 10 degrees) with a C2' endo/O1' endo sugar pucker (delta = 143 +/- 2 degrees), (-) synclinal about the C3'-O3' bond (epsilon = 273 +/- 4 degrees), trans, gauche about the C4'-C5' bond (gamma = 301 +/- 29 degrees) and either (-) or (+) clinal about the C5'-O5' bond (beta = 92 +/- 8 degrees or 274 +/- 3 degrees). The structure of 3',5'-pdTp in the crystalline complex differs due to rotations about the C4'-C5' bond (gamma = 186 +/- 12 degrees, gauche, trans) and the C5'-O5' bond [beta = 136 +/- 10 degrees, (+) anticlinal]. The undistorted conformation of enzyme-bound metal-3',5'-pdTp determined by NMR was docked into the X-ray structure of the enzyme, using 19 intermolecular NOEs from ring proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the inhibitor. van der Waals overlaps were then removed by energy minimization. Subsequent molecular dynamics and energy minimization produced no significant changes, indicating the structure to be in a global rather than in a local minimum. While the metal-coordinated 5'-phosphate of the NMR-docked structure of 3',5'-pdTp overlaps with that in the X-ray structure, and similarly receives bifunctional hydrogen bonds from both Arg-35 and Arg-87, the thymine, deoxyribose, and 3'-phosphate are significantly displaced from their positions in the X-ray structure, with the 3'-phosphate receiving hydrogen bonds from Lys-49 rather than from Lys-84 and Tyr-85. The repositioned thymine ring permits hydrogen bonding to the phenolic hydroxyl of Tyr-115.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1993

Mutational tests of the NMR-docked structure of the staphylococcal nuclease-metal-3',5'-pdTp complex.

In the X-ray structure of the staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5:183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3',5'-pdTp in the staphylococcal nuclease-metal-pdTp complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D.J., Serpersu, E.H., Gittis, A.G., Lattman, E.E., Mildvan, A.S. (preceding paper)]. In the NMR-docked structure, the 5'-phosphate of 3',5'-pdTp overlaps with that in the X-ray structure. However, the 3'-phosphate accepts a hydrogen bond from Lys-49 (2.89 A) rather than from Lys-84 (8.63 A), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 A) which does not occur in the X-ray structure (5.28 A). These interactions have been tested by binding studies of 3',5'-pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca2+ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While the K84A mutation did not significantly weaken 3',5'-pdTp binding to the enzyme (1.5 +/- 0.7 fold), the K49A mutation weakened 3',5'-pdTp binding to the enzyme by the factor of 4.4 +/- 1.8-fold. Similarly, the Y115A mutation weakened 3',5'-pdTp binding to the enzyme 3.6 +/- 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca(2+)-3',5'-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3',5'-pdTp than by the X-ray structure.

Binding Sites↗
PubMed1992

Two-dimensional NMR, circular dichroism, and fluorescence studies of PP-50, a synthetic ATP-binding peptide from the beta-subunit of mitochondrial ATP synthase.

PP-50, a peptide based on residues 141-190 of the beta-subunit of mitochondrial F1-ATPase, contains the GX4GKT consensus region for nucleoside triphosphate binding and has been shown to bind ATP [Garboczi, D.N., Shenbagamurthi, W.K., Hullihen, J., & Pedersen, P.L. (1988) J. Biol. Chem. 263, 812-816]. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (KD = 17.5 microM) with a 1:1 stoichiometry as shown by titrations measuring the partial quenching of ATP fluorescence by PP-50. CD spectra of PP-50 at pH 4.0 and at low ionic strength show 5.8% helix, 30.2% beta-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% beta-structure, and 48% coil. Increasing the ionic strength to 50 mM KCl also increases the structure, changing the CD to 7.4% helix, 64.4% beta-structure, and 28.2% coil. The 600-MHz proton NMR spectrum of PP-50, at pH 4.0 and low ionic strength, has been assigned by 2D methods (TOCSY, DQF-COSY, and NOESY with jump-return water suppression). Based on strong d alpha N NOEs, J alpha N values, and NH chemical shifts differing from random coil values, regions of extended structure are detected from residues 1-7 and 43-48. Based on dNN, dNN(i,i+2), and d alpha N(i,i+2) NOEs and 3J alpha N values, possible type I' and type I turns are found from residues 11-14 and 31-34, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction.

The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.

Bacterial Proteins↗
PubMed1992

Quantitative interpretations of double mutations of enzymes.

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 which kinetic or thermodynamic parameter of an enzyme is measured, the same two mutations can interact differently in the double mutant. Additive effects of two mutations on an equilibrium constant, such as the dissociation constant of the enzyme-substrate complex (KS), occur when noninteracting residues which facilitate the same step (substrate binding) are mutated. Partially additive effects result from the cooperative interaction with the substrate of the two residues mutated, and synergistic effects result from the anticooperative interaction with the substrate of the two residues mutated. An alternative explanation for synergy is extensive unfolding of the enzyme. Antagonistic effects on an equilibrium constant such as KS result from opposing structural effects of the two mutations on substrate binding. No additional effect of the second mutation in the double mutant represents a limiting case of either partial additivity or antagonism [corrected]. The interactions of the effects of two mutations on a rate constant such as kcat have the same explanations as those given above for equilibrium constants since the binding of a rate-limiting transition state is occurring. However, due to kinetic complexity, the following exceptions and additions exist. Additive effects of two mutations on kcat occur when noninteracting residues which facilitate the same step are mutated, provided this step is rate limiting. If the affected step is not rate limiting then synergistic effects of the two mutations are observed as each mutation causes the step to become progressively more rate limiting. Additive effects on kcat also occur when the two mutations affect consecutive steps, provided one of them is rate limiting. Partially additive effects on kcat also occur when noninteracting residues facilitating consecutive, non-rate-limiting steps are mutated. These concepts, when applied to published data on double mutants of delta 5-3-ketosteroid isomerase, staphylococcal nuclease, tyrosyl-tRNA synthetase, glutathione reductase, and subtilisin, provide deeper insights into the independent, cooperative, anticooperative, or antagonistic interactions of amino acid residues in the binding of substrates, activators, and inhibitors and in promoting catalysis.

Amino Acid Sequence↗
PubMed1992

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).

Binding, Competitive↗