Zinc interactions with regulatory dimers from Escherichia coli aspartate transcarbamoylase

Zn2+ is tetrahedrally bonded to the 4 nonadjacent thiols of each regulatory chain (Mr 17,000) near r-c contacts between catalytic (c) and regulatory chains (r) in aspartate transcarbamoylase (ATCase; c6r6). This paper reports on Zn2+ interactions with r dimer in the absence of stabilizing r-c contacts. After r2 and c3 subunits were separated, -SH groups of r2 were titrated with p-(hydroxymercuri)benzenesulfonate (PMPS) at pH 7.0. The concomitant release of Zn2+ (2 equiv/r dimer) was quantitated with 4-(2-pyridylazo)resorcinol (PAR) and was a linear function of PMPS added until 8 mercaptide bonds per r2 were formed. Breakage of 1 of 4 Zn2(+)-sulfur bonds in a Zn2+ binding cluster therefore makes the other three bonds more labile. From stopped-flow measurements, the PMPS-promoted Zn2+ release from r2 or mercaptide bond formation with 10- to 20-fold excess PMPS/r2-SH at pH 7.0 was first order with an Arrhenius activation energy Ea = 10 kcal/mol and a half-time t 1/2 = 9 +/- 2 ms at 20 degrees C without inhibitory anions present. The rate of mercurial-promoted Zn2+ release from r2 is at least 77 times faster than that from intact c6r6 [Hunt, J.B., Neece, S.H., Schachman, H.K., and Ginsburg, A. (1984) J. Biol. Chem. 259, 14793]; this indicates that Zn2+ binding clusters are more accessible to attack by PMPS than are those in ATCase. The addition of a 25-fold excess of the multidentate fluorescent chelator quin-2 to r2 gave a rate of Zn2+ dissociation that was 1/210th of that observed with excess mercurial. Furthermore, the Zn(PAR)1 complex was identified as the active species in the transfer of Zn2+ from Zn(PAR)2 to aporegulatory subunits, with kappa = (8 +/- 3) x 10(5) M-1 s-1 at pH 7.0 and 15 degrees C for this second-order association reaction. Although kinetic results are dependent on the mechanisms involved, an affinity constant K'A = (1.3 +/- 0.6) x 10(12) M-1 for Zn2+ binding to r dimer at pH 7.0 and 20 degrees C in the absence and presence of 100 mM KCl could be determined spectrally by rapid equilibration with the high-affinity, sensitive metalloindicators indo-1 and quin-2. This K'A value is based on the assumptions that Zn2+ binding sites in r2 are equivalent (noninteracting) and that apo-r2 does not dissociate; if apo-r2 dissociates, K'A approximately 10(14) M-1. Within experimental error, the K'A value was independent of [indo-1]/[r2] ratios from 36 to 3 with 0.3-8 microM r2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Zinc ions inhibit oxidation of cytochrome c oxidase by oxygen

Cytochrome c oxidase is a membrane-bound enzyme that catalyses the reduction of O2 to H2O and uses part of the energy released in this reaction to pump protons across the membrane. We have investigated the effect of addition of Zn2+ on the kinetics of two reaction steps in cytochrome c oxidase that are associated with proton pumping; the peroxy to oxo-ferryl (P(r)-->F) and the oxo-ferryl to oxidised (F-->O) transitions. The Zn2+ binding resulted in a decrease of the F-->O rate from 820 s(-1) (no Zn2+) to a saturating value of approximately 360 s(-1) with an apparent K(D) of approximately 2.6 microM. The P(r)-->F rate (approximately 10[(4) s(-1)] before addition of Zn2+) decreased more slowly with increasing Zn2+ concentration and a K(D) of approximately 120 microM was observed. The effects on both kinetic phases were fully reversible upon addition of EDTA. Since both the P(r)-->F and F-->O transitions are associated with proton uptake through the D-pathway, a Zn2+-binding site is likely to be located at the entry point of this pathway, where several carboxylates and histidine residues are found that may co-ordinate Zn2+.     

The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase

The metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) has been used at pH 7.0 to monitor the mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase and Zn2+ uptake by regulatory dimers upon displacement of the mercurial reagent with 2-mercaptoethanol. The release of Zn2+ (as reflected by a yellow to orange color change in PAR solutions) is linked to dissociation of the enzyme since the six Zn2+ bonding domains stabilize catalytic and regulatory chain contacts; the rebinding of Zn2+ produces enzyme assembly and a corresponding decrease in the amount of PAR-Zn2+ complex. Using greater than 10-fold PAR to free Zn2+ at pH 7.0, delta epsilon = 6.6 +/- 0.2 X 10(4) M-1 cm-1 at 500 nm (20 degrees C) for (PAR)2Zn2+ complex formation (beta'2 approximately equal to 10(12) M-1). In kinetic studies at pH 7.0, PAR (10(-4) M) has been used to measure the instantaneous concentration of Zn2+ released from micromolar quantities of protein; second-order k = 2 X 10(7) M-1 s-1 for forming the 1:1 PAR:Zn2+ complex. These properties of PAR-Zn2+ interactions make PAR a generally useful reagent for studying Zn2+ release from proteins.     

The circumsphere as a tool to assess distortion in [4Fe-4S] atom clusters

The geometry proposition that "four points not in a plane describe one and only one sphere" provides a novel tool for analyzing protein-induced distortions in [4Fe-4S] clusters. A geometrically perfect reference structure comprises interlaced, regular tetrahedra of Fe, S, and S gamma atoms having T(d) symmetry. Three circumspheres are defined by the three sets of four atoms, the circumcenters of which are unique points within the cluster. The structure is thus re-defined by the positions of the circumcenters in xyz space and the r, theta, phi of each atom on its respective sphere. Analysis of 12 high-resolution structures of protein-bound and small molecule [4Fe-4S](SR)(4) clusters revealed: (a) the circumcenters are generally non-coincident by approximately 0.01 to approximately 0.06 A; (b) the Fe radius, r(Fe), is nominally independent of core oxidation state, having values between 1.66 to 1.69 A, whereas r(S) and r(SG), which have ranges of 2.18-2.24 A and 3.87-3.94 A, respectively, both increase by as much as approximately 3% upon reduction from the 3+ to the 1+ core valence; (c) deviation of some atoms from the theta, phi of a perfect tetrahedron can be large, approximately 10 degrees, and sets of atoms can show patterns of motion on their spheres that result from changes in Fe-S bond lengths. Density functional theory calculations suggest that the [4Fe-4S] core itself requires rather little energy to distort (approximately 2 kcal/mol), whereas significantly more energy is required to distort the Sgamma shell (~4 kcal/mol) to that of cluster I in Clostridium acidurici ferredoxin.     

Two mechanisms of H+/OH- transport across phospholipid vesicular membrane facilitated by gramicidin A.

Two rate-limiting mechanisms have been proposed to explain the gramicidin channel facilitated decay of the pH difference across vesicular membrane (delta pH) in the pH region 6-8 and salt (MCI, M+ = K+, Na+) concentration range 50-300 mM. 1) At low pH conditions (approximately 6), H+ transport through the gramicidin channel predominantly limits the delta pH decay rate. 2) At higher pH conditions (approximately 7.5), transport of a deprotonated species (but not through the channel) predominantly limits the rate. The second mechanism has been suggested to be the hydroxyl ion propogation through water chains across the bilayer by hydrogen bond exchange. In both mechanisms alkali metal ion transport providing the compensating flux takes place through the gramicidin channels. Such an identification has been made from a detailed study of the delta pH decay rate as a function of 1) gramicidin concentration, 2) alkali metal ion concentration, 3) pH, 4) temperature, and 5) changes in the membrane order (by adding small amounts of chloroform to vesicle solutions). The apparent activation energy associated with the second mechanism (approximately 3.2 kcal/mol) is smaller than that associated with the first mechanism (approximately 12 kcal/mol). In these experiments, delta pH was created by temperature jump, and vesicles were prepared using soybean phospholipid or a mixture of 94% egg phosphatidylcholine and 6% phosphatidic acid.     

Role of protons in the thermodynamic contribution of a Zn(II)-Cys4 site toward metalloprotein stability

The current limited understanding of the free energy contributions of metal-protein interactions toward metalloprotein stability is largely due to an inability to separate the energetics of the metal-ligand and protein-protein interactions. In order to elucidate the thermodynamic contribution of a Zn(II)-(S.Cys)4 site toward metalloprotein stability relevant to classic structural Zn(II) sites, the reaction of {Zn(II)(H2O)6}2+ with a minimal, unstructured, tetracysteine 16-mer peptide, GGG, is described. Isothermal titration fluorimetry over the pH range of 4.5 to 9.0 is used to measure the free energy of Zn(II) binding to the model peptide GGG. The data show that, in the absence of proton competition, Zn(II) binds to the Cys4 coordination sphere with a Kd of 60 aM, indicating that the Zn(II)-(S.Cys)4 interaction can provide up to 22.1 kcal mol-1 in driving force for protein stabilization, folding, and/or assembly. Isothermal titration calorimetry shows that Zn(II)-GGG formation is entropy driven because of water release from both the metal and the peptide scaffold. At pH 7.0, where the Zn(II)-GGG Kd value is 8.0 pM, the reaction releases 3.8 protons, is endothermic with DeltaHrxn of +6.4 kcal mol-1, and entropy driven with DeltaSrxn of +72 cal K-1 mol-1. At pH 8.0, where the peptide is partially deprotonated prior to Zn(II) binding, the 1.0 fM Zn(II)-GGG Kd value reflects a Zn(II) complexation reaction involving the release of 2.5 protons, which is slightly exothermic, with DeltaHrxn of -2.0 kcal mol-1, and largely entropy driven, with DeltaSrxn of +61 cal K-1 mol-1. At pH 5.5, where proton competition weakens the Kd to 4.0 microM, only 3.2 protons are released upon Zn(II) binding, the reaction is endothermic, with DeltaHrxn of +7.7 kcal mol-1, and entropy driven, with DeltaSrxn of +51 cal K-1 mol-1. Likely an intrinsic property of Zn(II)-(S.Cys)4 sites, the entropy driven binding of Zn(II) reflects the proton dependent chemical speciation of the Zn(II)-(S.Cys)4 peptide complex and its effects on modulating the dehydration of both the peptide and metal. Furthermore, the Zn(II) binding thermodynamics of a variety of Zn(II) proteins at pH 7.0 reveals the presence of enthalpy-entropy compensation (EEC) phenomena in nature.     

Differential effects of zinc on native GABA(A) receptor function in rat hippocampus and cerebellum.

Hippocampal noradrenergic and cerebellar glutamatergic granule cell axon terminals possess GABA(A) receptors mediating enhancement of noradrenaline and glutamate release, respectively. The hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar one is not affected by benzodiazepine agonists, indicating the presence of an alpha6 subunit. We tested here the effects of Zn2+ on these two native GABA(A) receptor subtypes using superfused rat hippocampal and cerebellar synaptosomes. In the cerebellum, zinc ions strongly inhibited (IC50 approximately 1 microM) the potentiation of the K(+)-evoked [3H]D-aspartate release induced by GABA. In contrast, the GABA-evoked release of [3H]noradrenaline from hippocampal synaptosomes was much less sensitive to Zn2+ (IC50 > 30 microM). The effects of Zn2+ were then studied in two rat lines selected for high (ANT) and low (AT) alcohol sensitivity because granule cell GABA(A) receptors in ANT, but not AT, rats respond to benzodiazepine agonists due to a critical mutation in the alpha6 subunit. GABA increased the K(+)-evoked release of [3H]DCNS REGIONS-aspartate from cerebellar synaptosomes of AT and ANT rats, an effect prevented by the GABAA selective antagonist bicuculline. In AT rat cerebellum, the effect of GABA was strongly inhibited by Zn2+ (IC50 < or = 1 microM), whereas in ANT rats, the divalent cation was about 100-fold less potent. Thus, native benzodiazepine-sensitive GABAA receptors appear largely insensitive to functional inhibition by Zn2+ and vice versa. Changes in sensitivity to Zn2+ inhibition consequent to mutations in cerebellar granule cell GABA(A) receptor subunits may lead to changes in glutamate release from parallel fibers onto Purkinje cells and may play important roles in cerebellar dysfunctions.     

In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: implications for protein folding and assembly.

Because the N- and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild-type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16-21 mumol carbamoylaspartate per microgram per h, compared with 15 for wild-type ATCase, and the values for K0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild-type enzyme. As observed with wild-type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild-type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N- and C-termini exhibited the homotropic and heterotropic effects characteristic of wild-type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand-promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N- and C-termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.     

Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening

The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterotropic effectors promote a global conformational change in aspartate transcarbamoylase

The sigmoidal dependence of activity on substrate concentration exhibited by the regulatory enzyme aspartate transcarbamoylase (ATCase) of Escherichia coli is generally attributed to a ligand-promoted change in the quaternary structure of the enzyme. Although a global conformational change in ATCase upon the binding of ligands to some of the six active sites is well documented, a corresponding alteration in the structure of the wild-type enzyme upon the addition of the inhibitor, CTP, or the activator, ATP, has not been detected. Such evidence is essential for testing whether heterotropic, as well as homotropic, effects can be accounted for quantitatively in terms of coupled equilibria involving a conformational change in the enzyme and preferential binding of ligands to one conformation or the other. This evidence has now been obtained with a mutant form of ATCase in which Lys 143 in the regulatory chain was replaced by Ala, thereby perturbing interactions at the interface between the regulatory and catalytic chains in the enzyme and destabilizing the low-activity, compact (T) conformation relative to the high-activity, swollen (R) state. Difference sedimentation velocity experiments involving measurements of the changes caused by the binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate demonstrated that the sedimentation coefficient of the mutant enzyme was intermediate between that observed for the T and R states of wild-type ATCase. We interpret the results as indicating that the [T]/[R] ratio in phosphate buffer at pH 7.0 is reduced from about 2 X 10(2) for the wild-type enzyme to 2.7 for r143Ala ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase

Cytochrome c oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides CytcO with O2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn2+. The results show that addition of Zn2+ at concentrations of < or = 250 microM to the outside of the vesicles did not alter the transition rates between intermediates PR (P3)-->F3-->O4. However, proton pumping was impaired specifically during the P3-->F3, but not during the F3-->O4 transition at Zn2+ concentrations of < or = 25 microM. Furthermore, proton pumping during the P3-->F3 transition was typically impaired with the "as isolated" CytcO, which was found to contain Zn2+ ions at microM concentration. As has already been shown, Zn2+ was also found to obstruct proton uptake during the P3-->F3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a KI of approximately 1 microM for this binding site. In conclusion, the results show that Zn2+ ions bind on both sides of CytcO and that binding of Zn2+ at the proton output side selectively impairs proton release during the P3-->F3 transition.     

Cyclic nucleotides modulate the release of [3H] adenosine cyclic 3',5'-phosphate bound to the regulatory moiety of protein kinase I by the catalytic subunit of the kinase

The rate of release of bound c[3H]AMP from the two types (A and B) of cAMP binding sites on the regulatory subunit dimer (R2I) of rabbit muscle protein kinase I was studied in the presence of the catalytic (C) subunit of protein kinase. Rebinding of released c[3H]AMP was avoided by using highly diluted reactants or adding unlabeled cAMP or its analogues. No significant C-induced dissociation of R2I-(c[3H]AMP)4 occurred in the absence of Mg2+-ATP. Of the two options that one or two molecules of C are required to induce the release of c[3H]AMP bound to R2I, only the first one was compatible with the first-order dependence on [C] of the rate of release of c[3H]AMP observed over a wide range of C concentrations. In the absence of added unlabeled cyclic nucleotide, the rate of the C-induced release of c[3H]AMP was the same from site A and site B. The apparent second-order rate constant for the association of C to R2I(c[3H]AMP)4 was 6 X 10(6) M-1 s-1 (37 degrees C, 0.15 M KCl). Raising the concentration of unlabeled cAMP in the medium up to 1 microM decreased by up to 50% the rate of the C-induced release of bound c[3H]AMP from both sites. This is explained by assuming that the association of one molecule of C to R2I(c-[3H]AMP)4 leads to the release of c[3H]AMP first from one R subunit and subsequently, by a process that can be blocked by about 1 microM cAMP, from the other R subunit. A further rise of the cAMP concentration decreased the rate of release from site B only, so that the C-induced release of c[3H]AMP occurred almost exclusively from site A at very high concentrations of cAMP. This suggests that c[3H]AMP is released first from site A and that this vacant site by interacting with cAMP inhibits the release of c[3H]AMP from site B of the same R subunit. The role of site A in controlling the C-induced release was further supported by the finding that several cAMP analogues inhibited the release with potencies correlating with their affinities for site A. The C-induced release of c[3H]AMP from aged R2I was about 10 times slower than that from fresh R2I. No significant C-induced release of c[3H]AMP was observed from the monomeric fragment obtained by limited trypsin treatment of R2(1).     

A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

A kinetic scheme is presented for Lactobacillus casei dihydrofolate reductase that predicts steady-state kinetic parameters. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. Two major features of this kinetic scheme are the following: (i) product dissociation is the rate-limiting step for steady-state turnover at low pH and follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex; (ii) the rate constant for hydride transfer from NADPH to dihydrofolate (H2F) is rapid (khyd = 430 s-1), favorable (Keq = 290), and pH dependent (pKa = 6.0), reflecting ionization of a single group. Not only is this scheme identical in form with the Escherichia coli kinetic scheme [Fierke et al. (1987) Biochemistry 26, 4085] but moreover none of the rate constants vary by more than 40-fold despite there being less than 30% amino acid homology between the two enzymes. This similarity is consistent with their overall structural congruence. The role of Trp-21 of L. casei dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Trp-21, a strictly conserved residue near both the folate and coenzyme binding sites, was replaced by leucine. Two major effects of this substitution are on (i) the rate constant for hydride transfer which decreases 100-fold, becoming the rate-limiting step in steady-state turnover, and (ii) the affinities for NADPH and NADP+ which decrease by approximately 3.5 and approximately 0.5 kcal mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.     

Oxymyohemerythrin: discriminating between O2 release and autoxidation

Myohemerythrin (Mhr) is a non-heme iron O2 carrier (with two irons in the active site) that is typically found in the retractor muscle of marine 'peanut' worms. OxyMhr may either release O2, or undergo an autoxidation reaction in which hydrogen peroxide is released and diferric metMhr is produced. The autoxidation reaction can also be promoted by the addition of certain anions to Mhr solutions. This work, using recombinant Themiste zostericola Mhrs, contrasts the results of environmental effects on these reactions. For the O2 release reaction, deltaVdouble dagger(21.5 degrees C) = +28+/-3 cm3 mol(-1), deltaHdouble dagger(1 atm) = +22+/-1 kcal mol(-1), and deltaSdouble dagger(1 atm) = +28+/-4 eu. The autoxidation reaction (pH 8.0, 21.5 degrees C, 1 atm) displays different kinetic parameters: deltaVdouble dagger = -8+/-2 cm3 mol(-1), deltaHdouble dagger = +24.1+/-0.7 kcal mol(-1), and deltaSdouble dagger = +1+/-1 eu. Autoxidation in the presence of sodium azide is orders of magnitude faster than solvolytic autoxidation. The deltaVdouble dagger parameters for azide anation and azide-assisted autoxidation reaction are +15+/-2 and +59+/-2 cm3 mol(-1), respectively, indicating that the rate-limiting steps for the Mhr autoxidation and anation reactions (including O2 uptake) are not associated with ligand binding to the Fe2 center. The L103V and L103N oxyMhr mutants autoxidize approximately 10(3)-10(5) times faster than the wild-type protein, emphasizing the importance of leucine-103, which may function as a protein 'gate' in stabilizing bound dioxygen.     

An early transition state for folding of the P4-P6 RNA domain

Tertiary folding of the 160-nt P4-P6 domain of the Tetrahymena group I intron RNA involves burying of substantial surface area, providing a model for the folding of other large RNA domains involved in catalysis. Stopped-flow fluorescence was used to monitor the Mg2+-induced tertiary folding of pyrene-labeled P4-P6. At 35 degrees C with [Mg2+] approximately 10 mM, P4-P6 folds on the tens of milliseconds timescale with k(obs) = 15-31 s(-1). From these values, an activation free energy deltaG(double dagger) of approximately 8-16 kcal/mol is calculated, where the large range for deltaG(double dagger) arises from uncertainty in the pre-exponential factor relating k(obs) and delta G(double dagger). The folding rates of six mutant P4-P6 RNAs were measured and found to be similar to that of the wild-type RNA, in spite of significant thermodynamic destabilization or stabilization. The ratios of the kinetic and thermodynamic free energy changes phi = delta deltaG(double dagger)/delta deltaG(o') are approximately 0, implying a folding transition state in which most of the native-state tertiary contacts are not yet formed (an early folding transition state). The k(obs) depends on the Mg2+ concentration, and the initial slope of k(obs) versus [Mg2+] suggests that only approximately 1 Mg2+ ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg2+ ions. The observation of a substantial deltaG(double dagger) despite an early folding transition state suggests that a simple two-state folding diagram for Mg2+-induced P4-P6 folding is incomplete. Our kinetic data are some of the first to provide quantitative values for an activation barrier and location of a transition state for tertiary folding of an RNA domain.     

Temperature dependence of endocytosis mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. Evidence for two potentially rate-limiting steps

The rate of endocytosis of cell surface-bound [3H]-asialo-orosomucoid was determined as a function of temperature. Freshly isolated rat hepatocytes were allowed to bind [3H]asialo-orosomucoid at 4 degrees C, washed to remove nonbound ligand, and internalization was then assessed by the resistance of cell-associated radioactivity to release by the Ca2+ chelator EDTA. At 10 degrees C or below, endocytosis is negligible. Above 10 degrees C, the rate of endocytosis is proportional to temperature but the increase of the rate of endocytosis with increasing temperature changes sharply at about 20 degrees C. From 10-20 degrees C, the apparent activation energy for endocytosis, calculated from an Arrhenius plot, is 45.9 kcal/mol and the temperature coefficient, Q10, is 15.6. However, between 20 and 41 degrees C, the calculated activation energy is 17.0 kcal/mol and the Q10 is 2.6. Although the rate of endocytosis of previously bound [3H]asialo-orosomucoid is very dependent on the temperature, the final extent of endocytosis is essentially temperature-independent between 14 and 37 degrees C. The results suggest that there are at least two steps in the overall process of endocytosis mediated by the asialoglycoprotein receptor on isolated hepatocytes which can be potentially rate-limiting, one at 10 degrees C and another at approximately 20 degrees C.     

The Glu(B13) carboxylates of the insulin hexamer form a cage for Cd2+ and Ca2+ ions

Substitution of Cd2+ for Zn2+ yields a hexameric insulin species containing 3 mol of metal ion per hexamer. The Cd2+ binding loci consist of the two His(B10) sites and a new site involving the Glu(B13) residues located at the center of the hexamer [Sudmeier, J. L., Bell, S. J., Storm, M. C., & Dunn, M. F. (1981) Science (Washington, D.C.) 212, 560-562]. Substitution of Co2+ or Co3+ for Zn2+ gives hexamers containing 2 mol of metal per hexamer. Insulin solutions to which both Cd2+ and Co2+ have been added in a ratio of 6:2:1 [In]:[Co2+]:[Cd2+] followed by oxidation to the exchange-inert Co3+ state yield stable hybrid species containing both Co3+ and Cd2+ with a composition of (In)6(Co3+)2Cd2+. The kinetics of the reaction of 2,2',2"-terpyridine (terpy) with the exchange-labile (In)6(Cd2+)2 and (In)6(Co2+)2 derivatives are biphasic and involve the rapid formation of an intermediate with coordination of one terpy molecule to each protein-bound metal ion; then, in a rate-limiting step the terpy-coordinated metal ion dissociates from the protein, and a second molecule of terpy binds to the metal ion to form a bis complex. Reaction of the exchange-inert Co3+ ions of (In)6(Co3+)2 with terpy is a slow apparent first-order process (t1/2 = 13.1 h). In contrast to the kinetic behavior of (In)6(Co2+)2 and (In)6(Cd2+)2, the Cd2+ ions bound to the hybrid (In)6(Co3+)2Cd2+ react quite slowly with terpy (t1/2 = 1 h at pH 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel zinc snap motif conveys structural stability to 3-methyladenine DNA glycosylase I

The Escherichia coli 3-methyladenine DNA glycosylase I (TAG) is a DNA repair enzyme that excises 3-methyladenine in DNA and is the smallest member of the helix-hairpin-helix (HhH) superfamily of DNA glycosylases. Despite many studies over the last 25 years, there has been no suggestion that TAG was a metalloprotein. However, here we establish by heteronuclear NMR and other spectroscopic methods that TAG binds 1 eq of Zn2+ extremely tightly. A family of refined NMR structures shows that 4 conserved residues contributed from the amino- and carboxyl-terminal regions of TAG (Cys4, His17, His175, and Cys179) form a Zn2+ binding site. The Zn2+ ion serves to tether the otherwise unstructured amino- and carboxyl-terminal regions of TAG. We propose that this unexpected "zinc snap" motif in the TAG family (CX(12-17)HX(approximately 150)HX(3)C) serves to stabilize the HhH domain thereby mimicking the functional role of protein-protein interactions in larger HhH superfamily members.