Marvin W. Makinen

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods. Values of kcat and kcat/Km were 4.5 ± 0.1 s−1 and 0.49 ± 0.1 × 106 M−1⋅s−1 in the absence of divalent metal ions, 14.7 ± 2.2 s−1 and 25.1 ± 7.4 × 106 M−1⋅s−1 in the presence of Mg2+ or Mn2+, and 24.2 ± 3.6 s−1 and 2.4 ± 0.7 × 106 M−1⋅s−1 when supported by VO2+ or bis(acetylacetonato)oxovanadium(IV). Binding of VO2+ to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg2+. Electron paramagnetic resonance spectra of the ternary enzyme–VO2+-chelate–dUDP complex revealed a pattern of 31P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO2+ ion. Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced. By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO2+. We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO2+ and the VO2+-chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the C-terminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.

Structural Origins of the Insulin-mimetic Activity of Bis(acetylacetonato)oxovanadium(IV)

We have investigated the interaction of bis(acetylacetonato)oxovanadium(IV) (VO(acac)2) with bovine serum albumin (BSA) by EPR and angle-selected electron nuclear double resonance, correlating results with assays of glucose uptake by 3T3-L1 adipocytes. EPR spectra of VO(acac)2 showed no broadening in the presence of BSA; however, electron nuclear double resonance titrations of VO(acac)2 in the presence of BSA were indicative of adduct formation of VO(acac)2 with albumin of 1:1 stoichiometry. The influence of VO(acac)2 on uptake of 2-deoxy-d-[1-14C]glucose by serum-starved 3T3-L1 adipocytes was measured in the presence and absence of BSA. Glucose uptake was stimulated 9-fold in the presence of 0.5 mm VO(acac)2, 17-fold in the presence of 0.5 mm VO(acac)2 plus 1 mm BSA, and 22-fold in the presence of 100 nm insulin. BSA had no influence on glucose uptake, on the action of insulin, or on glucose uptake in the presence of VOSO4. The maximum insulin-mimetic effect of VO(acac)2 was observed at VO(acac)2:BSA ratios less than or equal to 1.0. Similar results were obtained also with bis(maltolato)oxovanadium(IV). These results suggest that the enhanced insulin-mimetic action of organic chelates of VO2+ may be dependent on adduct formation with BSA and possibly other serum transport proteins.

Determination of the Zero-Field Splitting Constants of High-Spin Metalloproteins by a Continuous-Wave Microwave Saturation Technique

Abstract A method is described for determining the zero-field splitting energy of high-spin metalloenzymes and proteins by a continuous-wave saturation technique with a conventional EPR spectrometer. The method requires analysis of graphical estimates of P 1 2 from saturation curves as a function of temperature. Under the condition that the signal amplitude depends only on the spin-lattice relaxation probability, the temperature dependence of P 1 2 provides an estimate of the zero-field splitting energy for spin-lattice relaxation via an Orbach process. The zero-field splitting energies of metmyoglobin and metmyoglobin fluoride are estimated at 22 and 12 cm−1, respectively, in excellent agreement with published results obtained by magnetic susceptibility and pulse saturation recovery methods. The zero-field splitting of methemoglobin is sensitive to the binding of inositol hexaphosphate. This change is shown to be consistent with the change in iron-porphyrin stereochemistry induced by binding of the organic phosphate molecule to the protein. The method can be applied to a wide variety of high-spin paramagnetic systems.

Catalytic conformation of carboxypeptidase A. The structure of a true reaction intermediate stabilized at subzero temperatures.

Abstract The structure of the mixed anhydride, acyl-enzyme intermediate of the esterolytic reaction of carboxypeptidase A is characterized by application of cryoenzymologic, magnetic resonance, and molecular graphics methods with use of the Co2+-substituted enzyme and the specific spin-label ester substrate O-3-(2,2,5,5-tetra-methylpyrrolinyl-1-oxyl)-propen-2-oyl- l -β-phenyllactate. A radial separation of 7·7 A between the active site Co2+ and the nitroxide group in the low temperature-stabilized acyl-enzyme intermediate is determined on the basis of their spin-spin (dipole-dipole) interactions. Application of molecular graphics techniques shows that the only configuration of the substrate that is sterically accommodated by the active site yields a calculated metal ion-to-nitroxide distance of 7·8 A. Steric accommodation of the spin-label in the active site requires severe torsional distortion around the aliphatic double bond of the propenoyl side-chain. Examination of the structure of the enzyme: spin-label intermediate reveals that the distortion arises from steric interactions of the pyrrolinyl group with the protein at a position that corresponds to the site occupied by the penultimate amide residue of an oligopeptide substrate from the site of cleavage. Together with kinetic data showing that hydrolysis of the spin-label is governed by rate-limiting deacylation, the results indicate that geometric distortion of substrates by secondary interactions with the enzyme, in general, is an obligatory part of the catalytic action of carboxypeptidase A. When viewed with respect to requirements for stereoelectronic control of bond cleavage in tetrahedral adducts of esters and amides (Deslongchamps, 1975) the results suggest that torsional distortion during catalysis results in rotation around the scissile bond of the substrate, and that this rotation is required to form the mixed anhydride reaction intermediate. These findings further support the interpretation that the hydrolysis of esters and amides catalyzed by carboxypeptidase A proceeds according to similar mechanisms except that formation of the mixed anhydride is rate-determining in peptide hydrolysis while deacylation of the mixed anhydride is rate-limiting in ester hydrolysis. Additionally, in this study application of the extension of the theory of the Solomon-Bloembergen-Morgan equations derived by Lindner (1965) for paramagnetic metal ions with S ≥ 1 demonstrates that the zero-field splitting of the high-spin Co2+ in the metal-substituted enzyme has no significant influence in determination of the relaxation enhancement of solvent protons by the active site metal ion.

ENDOR study of Gd3+ complexes in frozen solutions

Abstract The coordination environment of Gd3+ in frozen solutions is investigated by application of electron nuclear double-resonance spectroscopy. Proton ENDOR spectra of the Gd3+ ion in frozen methanol-water cosolvent mixtures obtained with the static laboratory magnetic field (Ha) at the turning point of the electron paramagnetic resonance absorption exhibit single-crystal-type line pairs. With use of selectively deuterated materials, the ligand origin of each pair of ENDOR lines has been assigned. For GdCl3 there are two distinguishable types of protons due to the HO groups of metal-coordinated solvent molecules, and there is one set belonging to the methyl group of metal-coordinated methanol. Similarly, for Gd(CH3000)3 and Gd(CH3CH2000)3, the set of ENDOR lines belonging to the methyl group of acetate and to the methylene group of propionate ligands have been identified. By analysis of the dependence of the ENDOR spectra on Ho, we have determined the values of the principal hyperfine coupling (hfc) components of each of the metal-bound ligands. The hfc components of methyl protons of Gd3+-bound acetate and of the methylene protons of Gd3+-bound propionate exhibit axial symmetry. Under the point-dipole approximation, they yield correspondingly calculated metal-proton distances of 4.53 and 4.42 A in good agreement with the value of 4.73 A deduced from crystallographic data for inner sphere coordinated ligands. The hfc components of the HO protons of metal-bound solvent molecules do not exhibit axial symmetry. One set is assigned to inner sphere coordinated H2O while the other is assigned to outer sphere bound CH30H. The metal-proton distances, calculated on the basis of the largest anisotropic hfc components as lower limit estimates, support these structural assignments. Application of ENDOR spectroscopy is made to identify the primary lanthanide binding site in a-chymotrypsin and to demonstrate the accuracy with which this method of analyzing ENDOR can be employed for structural characterization of metal complexes in frozen solutions. Comparison of the proton ENDOR spectrum of Gd3+ bound to a-chymotrypsin in frozen D20 solution to those of Gd(CH3000)3 and Gd(CH3CH2000)3 indicates that the lanthanide binding site in the protein includes a glutamate residue rather than an asparlate residue. Identification of this protein residue by ENDOR spectroscopy resolves discussions in the literature about the primary lanthanide binding site of a-chymotrypsin.

The effective position of the electronic point dipole of the nitroxyl group of spin labels determined by ENDOR spectroscopy

Abstract The effective position of the electronic point dipole of the nitroxyl group of the spin label 2.2,5,5-tetra methyl-l-oxypyrroline-3-carboxamide has been assigned by electron-nuclear double resonance and molecular modeling. From proton ENDOR spectra of the spin label in frozen solutions of perdeuterated dimethyl sulfoxide:chloroform: toluene (50:25: 25 v/v), we have identified the principal hyperfine coupling (hfc) components of the vinylic proton of the pyrrolinyl ring and of the two protons of the carboxamide side chain. The ENDOR-based dipolar He components are in excellent agreement with calculation when they are evaluated for each of the vinyl and carboxamide protons as the sum of two separate interactions with the nitroxyl nitrogen and oxygen atoms, weighted according to their respective EPR-assigned spin densities. Within experimental error, the corresponding electron-proton vectors belonging to the vinylic and the two carboxamide protons intersect on the NO bond. This intersection point is shown to be identical to that calculated as the effective position of the unpaired electron as a point dipole along the NO bond according to the EPR-determined fractional spin density associated with the nitrogen atom of the nitroxyl group.

ENDOR of Spin Labels for Structure Determination: From Small Molecules to Enzyme Reaction Intermediates

Electron paramagnetic resonance (EPR) is a sensitive method for probing the electronic and molecular structure of paramagnetic sites because the energies of microwave-induced transitions of unpaired electrons are modulated by interactions with nearby magnetic nuclei. This phenomenon is known as hyperfine (hf) interaction; measuring the strength of this interaction yields precise information about the electronic structure of the paramagnetic site and the geometric arrangement of magnetic nuclei in its immediate environment. While the most detailed information

A surfactant copolymer facilitates functional recovery of heat-denatured lysozyme.

Abstract: The triblock copolymer poloxamer 188 is a non‐cytotoxic, nonionic surfactant with both hydrophobic and hydrophilic domains. We show that P188 is able to facilitate the recovery of catalytic activity of heat‐denatured lysozyme in dilute solution at low molar ratios of P188:enzyme. Heat‐denatured enzyme retained 55% of native activity. After treatment with P188, the enzymes activity was 85% of native. Because of the low molar ratios used and the non‐cytotoxic nature of the compound, P188 may be of potential use in burn therapy.

Assignment of Proton Endor Resonances of Nitroxyl Spin-Labels in Frozen Solution

Spin-label nitroxyl derivatives of tetramethylpyrroline and tetramethylpyrrolidine in frozen solutions of perdeuterated methanol have been characterized by electron nucleus double resonance (ENDOR spectroscopy). With use of selectively deuterated derivatives of 2,2,5,5-tetramethylpyrroline-1-oxyl-3-carboxamide, proton ENDOR resonance features have been assigned to the vinylic proton in the five membered pyrrolinyl ring and to the methyl groups. The ENDOR resonance features were analyzed on the basis of their dependence on H0. Two pairs of resonance features were assigned to the vinylic proton and were shown to correspond to parallel and perpendicular hyperfine coupling (hfc) components. Six pairs of resonance features were ascribed to the methyl groups. The proton ENDOR spectra of the 3-carboxylic acid spin-label derivatives of tetramethylpyrroline and of tetramethylpyrrolidine compounds exhibited comparable features with nearly identical line splittings. From the observed ENDOR splittings, we have estimated the isotropic hfc component of the vinylic proton in 2,2,5,5-tetramethylpyrroline-1-oxyl-3-carboxamide to be -1.81 +/- 0.04 MHz in frozen methanol. On the basis of the anisotropic dipolar hfc components, the electron-to-vinylic proton distance is estimated as 3.78 +/- 0.01 A, in excellent agreement with that of 3.79 A calculated from X-ray defined coordinates.

The Second Derivative Electronic Absorption Spectrum of Cytochrome c Oxidase in the Soret Region

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3). Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.