Proteolytic modification of Escherichia coli alkaline phosphatase

Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.     

1H and 31P NMR studies of the binding of low-affinity anions to Cu,Zn superoxide dismutase

Anions that do not coordinate to the catalytically active copper ion of Cu,Zn superoxide dismutase, but still affect the activity of the enzyme by weaker interactions with the protein moiety surrounding the active site (low affinity anions), uniformly perturbed the 1H NMR line of the NH group of the copper ligand His 46. This effect was detected on the enzyme having Co(II) substituted for the native Zn(II), in which the resonances of residues bound to the copper are detected because of the antiferromagnetic coupling between Cu(II) and Co(II). The interaction with the enzyme of phosphate, a good representative of low-affinity anions, was also studied by 31P NMR of the native enzyme and of enzyme samples covalently modified at all lysines or at the Arg 141, which is 5 A away from the copper. The results obtained indicate that Arg 141 is a likely candidate for binding of low-affinity anions in the vicinity of the copper and that the 1H NMR line of His 46 NH is diagnostic for such an interaction.     

Zinc stoichiometry in Escherichia coli alkaline phosphatase. Studies by 31P NMR and ion-exchange chromatography.

31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

Phosphorylation and formation of hybrid enzyme species test the "half of sites" reactivity of Escherichia coli succinyl-CoA synthetase

Recent sequencing experiments have identified alpha-His246 as the phosphorylation site of Escherichia coli succinyl-CoA synthetase [Buck, D., Spencer, M. E., & Guest, J. R. (1985) Biochemistry 24, 6245-6252]. We have replaced alpha-His246 with an asparagine residue using site-directed mutagenesis techniques. The resulting mutant enzyme (designated H246N) exhibited no enzyme activity, as expected, but was found as a structurally intact, stable tetramer. Small differences in the net charge of H246N and wild-type enzymes were first detected on native polyacrylamide gels. These charge differences were resolved by using native isoelectric focusing gels to further separate the wild-type enzyme into diphosphorylated, monophosphorylated, and unphosphorylated species. The enzyme species were found to be interconvertible upon incubation with the appropriate enzyme substrate(s). Sample mixtures containing increasing molar ratios of H246N (alpha H246N beta)2 to wild-type enzyme (alpha beta)2 were unfolded and then refolded. The refolded enzyme mixtures were analyzed for enzymatic activity and separated on native isoelectric focusing gels. The hybrid enzyme (alpha beta alpha H246N beta) retained a significant amount of enzyme activity and also exhibited substrate synergism (stimulation of succinate in equilibrium succinyl-CoA exchange in the presence of ATP). Substrate synergism with this enzyme has been interpreted as evidence for interaction between active sites in such a way that only a single phosphoryl group is covalently attached to the enzyme at a given time [Wolodko, W. T., Brownie, E.R., O'Connor, M. D., & Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119]. On the contrary, we conclude that tetrameric succinyl-CoA synthetase from E. coli is comprised of two independently active dimer molecules associated together to form a "dimer of dimers" that displays substrate synergism within each dimer and not necessarily between dimers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hybridization studies of chicken liver fatty acid synthetase. Evidence for the participation in palmitate synthesis of cysteine and phosphopantetheine sulfhydryl groups on adjacent subunits

Enzymatically inactive variants of chicken liver fatty acid synthetase have been prepared by specific chemical modification of the active cysteine SH group with iodoacetamide, and the phosphopantetheine SH group with chloroacetyl-CoA. Hybridization of each of these variants with the unmodified enzyme yielded (modified)-(unmodified) hybrid dimers which possessed 50% synthetase activity. A 50% active (iodoacetamide-modified)-(chloroacetyl-CoA-modified) hybrid dimer was also demonstrated by recombination of these variants with each other. These results indicate that the two functional sites on the synthetase are independently active, and that each is comprised of a cysteine SH group from one subunit and a complementary phosphopantetheine SH group from the other subunit as depicted by the head-to-tail arrangement proposed by Wakil and co-workers (Wakil, S. J., Stoops, J. K., and Joshi, V.C.     

A hybrid Escherichia coli alkaline phosphatase formed on proteolysis

Cleavage of bacterial alkaline phosphatase by trypsin at the R-11, A-12 bond of both subunits results in changes in the structure and function of the enzyme as previously reported (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1985) J. Biol. Chem. 260, 7557-7561). A hybrid dimer has been formed by cleaving the R-11, A-12 bond of only one of the two subunits. This enzyme species has been purified and characterized to investigate subunit interactions of this hybrid dimeric enzyme species. Subunit interactions were observed using various methods to study functional and structural properties of the enzyme. In a kinetic study the T-2/A-12 hybrid enzyme was found to have a Vmax similar to the A-12 fully trypsin-modified enzyme. On exposure to EDTA the hybrid was found to lose activity at essentially the same rate as the A-12 enzyme presumably as a consequence of loss of metal ions required for function. On adding metal ions back to the apoenzyme form, activity of the hybrid was reconstituted to a degree similar to that of the native enzyme whereas the activity of the A-12 enzyme was reconstituted to a much lesser extent. The Tm of the hybrid measured by differential scanning calorimetry was closer to the value obtained for the A-12 enzyme than the T-2 enzyme but circular dichroic spectra indicated secondary structural features of the hybrid different from both symmetrical forms of the enzyme. These results provide evidence for strong subunit interactions in the alkaline phosphatase dimer.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR

Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.     

Crystal structure of peroxynitrite-modified bovine Cu,Zn superoxide dismutase.

The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing three-dimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160, 181-217, 1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr-108 of a second dimer.     

Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase

The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.     

Is the subunit the minimal function unit of creatine kinase?

The dimeric rabbit muscle isozyme of creatine kinase (MM) is modified by iodoacetamide to produce the inactive dimer (M'M') and then hybridized with native dimeric brain isozyme (BB). The hybrid enzyme (M'B), as isolated by PAGE, has the same Km for both ATP and creatine but half the specific activity of the brain isozyme (BB). Likewise, the hybrid of the modified brain with the native muscle isozyme (MB') has half the activity of the native muscle enzyme. The M'B, MB' and MB hybrid dimers all have essentially the same electrophoretic properties, and their intrinsic fluorescence and CD spectra in the far-ultraviolet region are very similar to those of the homodimers MM and BB. Similar results were obtained for the hybrid (M"B) containing the muscle enzyme subunit modified at both the thiol group with iodoacetamide and the Trp residue with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide and the native brain enzyme submit. The above results suggest strongly the independent catalytic function of the subunit of creatine kinase.     

Sequential binding and sensing of Zn(II) by Bacillus subtilis Zur

Bacillus subtilis Zur (BsZur) represses high-affinity zinc-uptake systems and alternative ribosomal proteins in response to zinc replete conditions. Sequence alignments and structural studies of related Fur family proteins suggest that BsZur may contain three zinc-binding sites (sites 1-3). Mutational analyses confirm the essential structural role of site 1, while mutants affected in sites 2 and 3 retain partial repressor function. Purified BsZur binds a maximum of two Zn(II) per monomer at site 1 and site 2. Site 3 residues are important for dimerization, but do not directly bind Zn(II). Analyses of metal-binding affinities reveals negative cooperativity between the two site 2 binding events in each dimer. DNA-binding studies indicate that BsZur is sequentially activated from an inactive dimer (Zur(2):Zn(2)) to a partially active asymmetric dimer (Zur(2):Zn(3)), and finally to the fully zinc-loaded active form (Zur(2):Zn(4)). BsZur with a C84S mutation in site 2 forms a Zur(2):Zn(3) form with normal metal- and DNA-binding affinities but is impaired in formation of the Zur(2):Zn(4) high affinity DNA-binding state. This mutant retains partial repressor activity in vivo, thereby supporting a model in which stepwise activation by zinc serves to broaden the physiological response to a wider range of metal concentrations.     

Limited proteolysis of human von Willebrand factor by Staphylococcus aureus V-8 protease: isolation and partial characterization of a platelet-binding domain

Purified human von Willebrand factor (vWF) was digested with Staphylococcus aureus V-8 protease, and specific domains interacting with platelets were isolated and characterized. Amino acid sequence analysis and sodium dodecyl sulfate gel electrophoresis demonstrated that the digestion proceeded primarily by a single cleavage of the native 270K subunit between an internal Glu-Glu peptide bond. This produced an integral stepwise degradation of the multimers of vWF with a concomitant accumulation of bands with mobility similar to that of the smaller molecular weight vWF multimers. The immediate precursor of the final products contained equimolar amounts of 270K subunit and of two polypeptides (170K and 110K). The cleavage of the remaining 270K subunit converted vWF into two main fragments (fragments II and III). These fragments were isolated by ion exchange chromatography, characterized, and assayed for platelet binding in the presence of ristocetin. Fragment III is a dimer of 315K composed primarily of two chains of 170K. Amino acid sequence analysis indicated that it originated from the amino-terminal portion of the 270K subunit and contained 11% of the original ristocetin cofactor activity. Also, it binds to platelets at the same specific sites as native vWF and shows a platelet binding pattern similar to that of partially reduced vWF (500K). Fragment II is a dimer of 235K composed of two identical chains of 110K. Amino acid sequence analysis indicated that it originated from the carboxyl-terminal portion of the 270K subunit and lacked ristocetin cofactor activity. Also, it does not bind to platelets or inhibit the binding of 125I-vWF in the presence of ristocetin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The refolding of lactate dehydrogenase subunits and their assembly to the functional tetramer.

The renaturation process of different lactate dehydrogenase isozymes (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from their unfolded subunits was investigated using a number of techniques. (a) kinetics of activity regain, (b) the kinetics of fluorescence change of fluoresecence change of the protein tryptophans, (c) kinetics of regain of the fluorescence properties of a covalently attached fluorescence probe (fluorescein) and (d) the kinetics of assembly, by following the intermediate oligomeric species appearing in the assembly pathway from monomers to tetramers. The results indicate that the unfolded polypeptide is converted to the active oligomeric species by the following scheme: Denatured subunit I leads to partially refolded subunit II leads to folded subunit III leads to dimer IV leads to tetramer. Step I and step II are first-order where step II is rate limiting. The ligands NAD+ and NADH accelerate step II, thus converting step I to the rate-limiting process. The fact that partially folded lactate dehydrogenase subunits are capable of co-enzyme binding may indicate the possible role of these ligands in the assembly of lactate dehydrogenase in vivo. Steps III and IV were found to be fast. The intermediate formation of an enzyme dimer which then dimerizes to the tetrameric species is found to be the major assembly pathway. Only a small portion of the lactate dehydrogenase tetramer is formed through the intermediate formation of a trimer intermediate.     

The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.     

The metal specificity and selectivity of ZntA from Escherichia coli using the acylphosphate intermediate

ZntA from Escherichia coli is a P-type ATPase that confers resistance to Pb(II), Zn(II), and Cd(II) in vivo. We had previously shown that purified ZntA shows ATP hydrolysis activity with the metal ions Pb(II), Zn(II), and Cd(II). In this study, we utilized the acylphosphate formation activity of ZntA to further investigate the substrate specificity of ZntA. The site of phosphorylation was Asp-436, as expected from sequence alignments. We show that in addition to Pb(II), Zn(II), and Cd(II), ZntA is active with Ni(II), Co(II), and Cu(II), but not with Cu(I) and Ag(I). Thus, ZntA is specific for a broad range of divalent soft metal ions. The activities with Ni(II), Co(II), and Cu(II) are extremely low; the activities with these non-physiological substrates are 10-20-fold lower compared with the values obtained with Pb(II), Zn(II), and Cd(II). Similar results were obtained with DeltaN-ZntA, a ZntA derivative lacking the amino-terminal metal binding domain. By characterizing the acylphosphate formation reaction in ZntA in detail, we show that a step prior to enzyme phosphorylation, most likely the metal ion binding step, is the slow step in the reaction mechanism in ZntA. The low activities with Ni(II), Co(II), and Cu(II) are because of a further decrease in the rate of binding of these metal ions. Thus, metal ion selectivity in ZntA and possibly other P1-type ATPases is based on the charge and the ligand preference of particular metal ions but not on their size.     

Binding and conformational analysis of phosphoramidate-restriction enzyme interactions

Phosphoramidates are modified deoxyoligonucleotides that feature nitrogen in place of the 3'-oxygen of a phosphodiester linkage. Noted for stability against nuclease activity, these linkages are of both mechanistic and therapeutic interest. While a number of studies characterizing the properties of oligonucleotides composed entirely of phosphoramidate linkages have been published, little is known about how singly substituted phosphoramidate substitutions affect the thermodynamics and structure of protein-oligonucleotide interactions. We chose to investigate these interactions with PvuII endonuclease, the DNA binding behavior of which is well-characterized. Oligonucleotide duplexes containing a phosphoramidate substitution at the scissile phosphates were resistant to cleavage by the enzyme, even after extended incubations. However, the enzyme was able to cleave the native strand in a native:phosphoramidate heteroduplex at a rate comparable to that observed with the native substrate. Ca(II)-stimulated PvuII binding for a phosphoramidate-substituted oligonucleotide is comparable to that of the native duplex (K(d) approximately 200 pM). K(d) values obtained in the presence of Mg(II) are somewhat weaker (K(d) approximately 10 nM). Under metal-free conditions, the enzyme exhibited a remarkable approximately 50-fold greater affinity for the modified oligonucleotide relative to the native substrate (5 vs 240 nM). While (31)P NMR spectra indicate increased chemical shift dispersion in the free phosphoramidate duplex, the spectrum of the enzyme-bound duplex is similar to that of the native duplex. (1)H-(15)N HSQC analysis indicates that enzyme conformations in the presence of these oligonucleotides are also comparable. The tight binding of the phosphoramidate duplex under metal-free conditions and its resistance to cleavage are attributed to local conformational adjustments propagating from the O-->N substitution.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Zinc (II) coordination in Escherichia coli DNA topoisomerase I is required for cleavable complex formation with DNA.

Escherichia coli DNA topoisomerase I catalyzes relaxation of negatively supercoiled DNA. The reaction proceeds through a covalent intermediate, the cleavable complex, in which the DNA is cleaved and the enzyme is linked to the DNA via a phosphotyrosine linkage. Each molecule of E. coli DNA topoisomerase I has been shown to have three tightly bound zinc(II) ions required for relaxation activity (Tse-Dinh, Y.-C., and Beran-Steed, R.K. (1988) J. Biol. Chem. 263, 15857-15859). It is shown here that Cd(II) could replace Zn(II) in reconstitution of active enzyme from apoprotein. The role of metal was analyzed by studying the partial reactions. The apoenzyme was deficient in sodium dodecyl sulfate-induced cleavage of supercoiled PM2 phage DNA. Formation of covalent complex with linear single-stranded DNA was also reduced in the absence of metal. However, the cleavage of small oligonucleotide was not affected, and the apoenzyme could religate the covalently bound oligonucleotide to another DNA molecule. Assay of noncovalent complex formation by retention of 5'-labeled DNA on filters showed that the apoenzyme was not inhibited in noncovalent binding to DNA. It is proposed that zinc(II) coordination in E. coli DNA topoisomerase I is required for the transition of the noncovalent complex with DNA to the cleavable state.