Temperature dependent conformational changes at the active site of pyruvate kinase. A li nuclear magnetic resonance study.

The interaction of Li⁺, a weak activator of pyruvate kinase, with substrate and inhibitor complexes of the enzyme has been investigated by magnetic resonance techniques. Proton relaxation rate (PRR) titrations indicate that the dissociation constant of Li⁺ from the ternary enzyme-Mn(II)-phosphoenolpyruvate (P-enolpyruvate) complex is 15 mm at 5 °C and 17 mm at 30 °C. The electron paramagnetic resonance spectrum of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex is the superposition of spectra for two distinct species (Reed, G. H., and Cohn, M. (1973) J. Biol. Chem.248, 6436–6442). Low temperatures favor the form giving rise to the more nearly isotropic spectrum, whereas high temperatures favor the species giving rise to the anisotropic “K⁺-like” spectrum. ⁷Li nuclear magnetic resonance data are consistent with a model in which the two forms observed by epr correspond to differing Mn(II) to Li(I) distances. The form giving rise to the anisotropic spectrum is characterized by a Mn(II) to Li(I) distance of 4.7 Å, and in the more isotropic form this distance is approximately 9 Å. The 4.7 Å separation of the Mn(II) and Li(I) in the anisotropic form of the complex compares favorably with the 4.9 Å separation of Mn(II) and T1(I) (Reuben, J., and Kayne, F. J. (1971) J. Biol. Chem.246, 6227–6234) in the P-enolpyruvate complex, although T1⁺ is a much better activator of the pyruvate kinase reaction. Thus, a change in the distance between the monovalent and divalent cations does not account quantitatively for the lower activation by Li⁺, inasmuch as more than 50% of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex has the “active” conformation with respect to the separation of the cations and the epr spectrum of the complex. As reported previously (Reed, G. H., and Morgan, S. D. (1974) Biochemistry13, 3537–3541), the dissociation constant of oxalate and the epr spectrum for the ternary complex of pyruvate kinase with Mn(II) and oxalate are not influenced by the species of monovalent cation present. The nuclear relaxation rates of Li⁺ are increased in the presence of the ternary oxalate complex, although the separation of the Mn(II) and Li(I) appears to be much greater than for the “anisotropic” form of the P-enolpyruvate complex.     

Phosphorus-containing purines and pyrimidines: A new class of transition state analogs

Synthetic routes to the [1,5,2]-diazaphosphorine (“4-phosphapyrimidine”), imidazo[4,5-e][1,5,2]-diazaphosphorine (“6-phosphapurine”), and imidazo[4,5-d][1,3,2]-diazaphosphorine (“2-phosphapurine”) ring systems have been developed. Appropriately functionalized derivatives of these heterocycles are desired as possible transition state analogs of the nucleoside deaminases.     

GuHC1 induced unfolding-folding transition of a hinge-bending protein: horse muscle phosphoglycerate kinase

The antineoplastic compound N2-methyl-9-hydroxyellipticinium (9-OH-NME) is able to bind to different biological molecules after an oxidative activation by horseradish peroxidase and hydrogen peroxide. In this study, the efficient covalent binding in vitro of 9-OH-NME onto RNA and poly A is described. The phenomenon is analyzed by different HPLC methods and the yield of binding is determined using [3H]9-OH-NME. For an initial ratio drug per nucleotide of 0.07, the rb obtained (ratio of drug bound per nucleotide) of 0.026 for RNA and 0.044 for poly A, which represent respectively a yield of 40% and 60% for the drug fixation onto these macromolecules. These facts demonstrate the high electrophilicity of para-quinone-imine derivatives in ellipticinium series.     

Thermodynamics and kinetics of co-operative protein-nucleic acid binding: II. Studies on the binding between protamine and calf thymus DNA

Unspecific binding of a protamine, namely fluorescein-labelled clupeine Z, to double-stranded calf thymus DNA was studied using fluorescence titration methods and chemical relaxation techniques. Both equilibrium and kinetic data have been analysed using general theoretical approaches discussed in the accompanying paper. The results agree well with the predictions made on the basis of a standard co-operative binding model.Basic parameters evaluated are the co-operative binding constant (K), the coefficient measuring co-operative interaction between nearest neighbours (q), the number of nucleotides occupied by one protamine molecule (n) and the rate constant of dissociation at the ends of bound ligand sequences (K_D). Values obtained at 20 °C, pH 7.5 and 0.4 m-NaCl were K = 5.8 × 10⁷m^?1, q = 1700, n = 20 and K_D = 0.29 s^?1. They have been found to be sensitive to the concentration of added salt (NaCl). This effect apparently reflects the essentially electrostatic nature of the binding process. The results can be satisfactorily described in terms of competitive binding of sodium ions.     

Complexation of Pd(II) with nucleosides. Evidence for a strong interaction Pd⋯HN by NMR

Bis-derivatives of phenylantimony(III) with some monothio-β-diketones have been synthesized and characterized as five coordination species by elemental analyses, molecular weight and spectral data. The stereochemistry of the complexes having asymmetrical ligands is discussed.     

Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases.

Five sensitive substrates of human leukocyte and porcine pancreatic elastase having the sequence MeO-Suc-Ala-Ala-Pro-Val-X where -X is -NA (4-nitroanilide), -SBzl (thiobenzyl ester), -OEt, -AMC (4-methyl-7-coumarylamide), or -NNapOMe (1-methoxy-3-naphthylamide), were synthesized. The kinetic constants for the enzymatic hydrolysis as well as the sensitivity of each substrate are reported. Hydrolysis of the peptide -AMC and -NNapOMe derivatives were followed by monitoring spectrofluorometrically the release of H-AMC and H-NNapOMe, respectively. Cleavage of the thiobenzyl ester yields benzyl mercaptan as the hydrolysis product. Its release was monitored at 412 nm by reaction with Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid)] in the assay mixture to produce the 3-carboxy-4-nitrothiophenoxide anion or at 324 nm by reaction with 4,4′-dithiodipyridine to produce 4-thiopyridone. Hydrolysis of the ethyl ester was measured using a coupled assay with NAD⁺ and liver alcohol dehydrogenase. The NADH⁺ formed upon oxidation of the ethanol released from cleavage of the ester was followed at 340 nm. MeO-Suc-Ala-Ala-Pro-Val-SBzl, the best substrate of the series, was capable of detecting as little as 2.4 pm (0.072 ng/ml) of active-site titrated human leukocyte elastase and 5.8 pm (0.15 ng/ml) of active-site titrated porcine pancreatic elastase using 4,4′-dithiodipyridine. The corresponding values with Ellman's reagent were 5.0 pm (0.15 ng/ml) and 7.4 pm (0.19 ng/ml), respectively. Advantages of this substrate are its high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values and ease of synthesis. One disadvantage is the interference of high concentration of thiols with the assay. The peptidyl-AMC is almost as sensitive as the thiobenzyl ester and can detect 11 pm of HL elastase and 18 pm of PP elastase. An advantage of this substrate is the fact that cleavage involves a peptide bond. Disadvantages are relatively low $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values and greater difficulty in synthesis. The peptidyl-NNapOMe has possible utility in histochemical studies due to the low intrinsic fluorescence of the substrate relative to the peptidyl-AMC. For a rate assay it has a lower sensitivity than either the thiobenzyl ester or peptidyl-AMC. The coupled liver alcohol dehydrogenase-ethyl ester assay offers no advantages except in cases where the ester substrate is commercially available. The 4-nitroanilide assay enjoys moderate sensitivity and is extremely convenient for routine use. Except for the peptidyl ethyl ester assay, all of the human leukocyte elastase assays reported in this paper are vastly more sensitive than any other existing assay for this protease.     

The complexation of transition series metal ions by nalidixic acid

The following formation constants have been determine for nalidixic acid: proton, copper(II) complexation, magnesium(II) complexation, guanosine-5′-monophosphate-copper(II) complexation. Use of these data (together with the corresponding published constants of calcium(II), iron(II), manganese(II) and zinc(II) supports the hypothesis that the drug acts at a site other than extracellular. Complex formation between nalidixic acid, metal ion and DNA (at guanosine residues) is suggested.     

Assembly-dependent conformational changes in a viral capsid protein. Calorimetric comparison of successive conformational states of the gp23 surface lattice of bacteriophage T4

Inter- and intra-subunit bonding within the surface lattice of the capsid of bacteriophage T4 has been investigated by differential scanning calorimetry of polyheads, in conjunction with electron microscopy, limited proteolysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bonding changes corresponding to successive stages of assembly of the major capsid protein gp23, including its maturation cleavage, were similarly characterized. The uncleaved/unexpanded surface lattice exhibits two endothermic transitions. The minor event, at 46 degrees C, does not visibly affect the surface lattice morphology and probably represents denaturation of the N-terminal domain of gp23. The major endotherm, at 65 degrees C, represents denaturation of the gp23 polymers. Soluble gp23 from dissociated polyheads is extremely unstable and exhibits no endotherm. Cleavage of gp23 to gp23* and the ensuing expansion transformation effects a major stabilization of the surface lattice of polyheads, with single endotherms whose melting temperatures (t*m) range from 73 to 81 degrees C, depending upon the mutant used and the fraction of gp23 that is cleaved to gp23* prior to expansion. Binding of the accessory proteins soc and hoc further modulates the thermograms of cleaved/expanded polyheads, and their effects are additive. hoc binding confers a new minor endotherm at 68 degrees C corresponding to at least partial denaturation of hoc. Denatured hoc nevertheless remains associated with the surface lattice, although in an altered, protease-sensitive state which correlates with delocalization of hoc subunits visualized in filtered images. While hoc binding has little effect on the thermal stability of the gp23* matrix, soc binding further stabilizes the surface lattice (delta Hd approximately +50%; delta t*m = +5.5 degrees C). It is remarkable that in all states of the surface lattice, the inter- and intra-subunit bonding configurations of gp23 appear to be co-ordinated to be of similar thermal stability. Thermodynamically, the expansion transformation is characterized by delta H much less than 0; delta Cp approximately 0, suggesting enhancement of van der Waals' and/or H-bonding interactions, together with an increased exposure to solvent of hydrophobic residues of gp23* in the expanded state. These findings illuminate hypotheses of capsid assembly based on conformational properties of gp23: inter alia, they indicate a role for the N-terminal portion of gp23 in regulating polymerization, and force a reappraisal of models of capsid swelling based on the swivelling of conserved domains.     

A scanning dual wavelength spectrophotometer: Application to the study of photosynthetic electron transport

A dual wavelength scanning spectrophotometric method for the analysis of small absorbance changes in turbid suspensions is presented, which improves upon the point by point method of obtaining difference spectra in terms both of accuracy and rapidity. Examples are shown of the application of this instrument, in conjunction with a computer curve-resolving routine, to problems in photosynthetic electron transport.     

Binding of terbium to apoferritin: a fluorescence study

Luminescence measurements show that apoferritin binds three Tb(III) atoms per subunit in accordance with crystallographic evidence. Fe(II) competes with Tb(III) for at least some of the binding sites. This competition may be the molecular basis for the inhibition of iron incorporation into apoferritin brought about by Tb(III). Ca(II), which is generally replaced by Tb(III) in Ca(II) binding proteins, does not compete with the lanthanide for binding to apoferritin.     

A kinetic and spectral study of the alkaline transitions of chloroperoxidase

The optical spectrum of chloroperoxidase in the near ultraviolet and visible region was studied from pH 6 to 12. Chloroperoxidase undergoes a first transition which is irreversible at pH 7 and a second transition near pH 11. The second transition is reversible provided the incubation period above pH 11 is kept as short as possible. The spectral properties of the intermediates were studied in the Soret region by means of a rapid scan apparatus. The rates of the transitions were measured in a stopped-flow apparatus. The pH dependence of both the spectra and the rate constants indicate that at least three ionizations are involved in the first alkaline transition.     

The conformational analysis of oligosaccharides by H-NMR and HSEA calculation

The application of 1H-nuclear Overhauser enhancement, 1H-spin-lattice-relaxation-time and 1H-chemical shift measurements for the assessment of the conformational preferences of oligosaccharides are briefly reviewed. It is demonstrated that additivity rules, for the correlation of the chemical shifts of similar hydrogen atoms in different oligosaccharides, can be useful in the conformational analysis of oligosaccharides when the differential chemical shifts are greater than 0.1 ppm. These often can be attributed to specific interunit deshielding of a hydrogen atom by an oxygen atom with which it is in strong nonbonded interaction. HSEA calculations are used to demonstrate that differential chemical shifts of less than 0.1 ppm can have origins that are not significant to the overall conformational preferences of the oligosaccharides which are being compared. Both shielding and deshielding effects can arise from a change in the orientation of a substituent group as the result of the introduction of a sugar on a neighboring unit. It is demonstrated that substituent groups, such as hydroxymethyl and acetamido groups, on occasions, should be treated in HSEA calculations as freely rotating about their linkage to a pyranose ring.     

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: A comparison and immunological study of purified solubilized preparations,and alteration of enzyme levels by cholestyramine feeding

Some properties of various preparations of solubilized 3-hydroxy-3-methylglutaryl CoA reductase from rat liver are described. One, prepared by solubilization with deoxycholate, has been brought to a level of purity such that only a sińgle component is detected by polyacrylamide gel electrophoresis. A second preparation, solubilized by high salt concentration and heat treatment, has also been purified to a high level of purity so that only minor contaminants are detected. The deoxycholate-solubilized 3-hydroxy-3-methylglutaryl CoA reductase has a molecular weight of 197,000–202,000. Electrophoresis of both preparations treated with mercaptoethanol on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed one band with a molecular weight of 65,000. The data are consistent with the trimeric structure consisting of three polypeptide chains of apparently identical molecular weight. An antiserum to the deoxycholate-solubilized preparation has been prepared. Despite major differences among these preparations in specific activity, in stability to cold, and in the requirement of high salt concentration for preservation, both samples react in the same manner to the antibody and are immunologically indistinguishable. A preparation solubilized by freeze-thawing also reacts with the antiserum. Possible reasons for the variations in specific activity are considered, and it is concluded that specific activity changes cannot be reliably related to protein concentration unless the protein is isolated.Application of the immunological assay to an analysis of the effect of feeding cholestyramine to rats shows that compared to normals the diurnal cycle is unchanged but the rate of enzyme protein synthesis in the cholestyramine-fed rats is greatly accelerated. However, the first-order rate constant for degradatation of enzyme protein remains essentially unchanged throughout the falling phases of the cycle. The specific activity relationships of the enzyme protein of cholestyramine-fed rats appear to be altered when compared to that of normally fed controls.     

Electronic effects in transition metal porphyrins. 5. Thermodynamics of axial ligand addition and spectroscopic trends of a series of symmetrical and unsymmetrical derivatives of tetraphenylporphinatozinc(II)

The electronic spectra and equilibrium constants for addition of 3-picoline to a series of symmetrically and unsymmetrically phenyl-substituted ZnTPP derivatives have been measured. It is found that the α band energy varies slightly nonlinearly with the sum of the Hammett sigma constants of the substituents within the series (p-Cl)_x(p-NEt₂)_yTPPZn(II), while the smaller variation in the β band appears to be linear. The log of the intensity ratio of the α and β bands, log A_β/A^?_α, however, varies linearly with the band energies of both the α and β bands for both 4- and 5-coordinate complexes of unsymmetrical as well as symmetrical ZnTPP derivatives. Likewise, log K_eq for 3-picoline addition varies linearly with the sum of the Hammett sigma constants for all complexes investigated. Thus the electronic effects of unsymmetrically placed substituents are averaged by the metal Zn to yield a Lewis acid strength toward 3-picoline which is dependent only upon the sum of the electronic effects and not on the identity of the substituents or the symmetry of their distribution.     

Transferase-deficiency galactosemia: evidence for the lack of a transferase protein in galactosemic red cells

Red blood cell lysates from normal individuals, a homozygous Duarte variant, and a patient with transferase-deficiency galactosemia were challenged with rabbit antibody to pure human placental galactose-1-phosphate uridylyltransferase. Although the antibody quantitatively precipitated the enzymatically active proteins in the normal and Duarte hemolysates, the Duarte sample absorbed only about one-half as much antibody as did the normal. In contrast, the antibody did not react with the galactosemic hemolysate.