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M W Makinen

Publications and source records attributed to M W Makinen.

At least 19 recordsLinked to original sources
PubMed1991

The pH variation of steady-state kinetic parameters of site-specific Co(2+)-reconstituted liver alcohol dehydrogenase. A mechanistic probe for the assignment of metal-linked ionizations.

To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1), the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters kcat and kcat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co(2+)-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell-shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing kcat (pK1 congruent to 6.7, pK2 congruent to 10.6) belong to the ternary enzyme-NAD(+)-alcohol complex, whereas the ionizations governing kcat/KM (pK1' congruent to 7.5, pK2' congruent to 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1' are not influenced by metal substitution and are ascribed to His-51 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2' are significantly decreased (delta pKa congruent to 1.0) in the Co(2+)-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD(+)-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.

1-Propanol↗
PubMed1990

Structure and conformation of the nitroxyl spin-label ethyl 3-(2,2,5,5-tetramethylpyrrolinyl-1-oxyl)-propen-2-oate determined by electron nuclear double resonance: comparison with the structure of a spin-label substrate of carboxypeptidase A.

The conformation of the nitroxyl spin-label ethyl 3-(2,2,5,5-tetramethylpyrrolinyl-1-oxyl)-propen-2-oate has been determined by electron nuclear double resonance (ENDOR) spectroscopy and computer-based molecular modeling. From ENDOR spectra of the compound in frozen solution, we have assigned resonance absorption features for each class of protons, and we have identified their principal hyperfine coupling (hfc) components from analysis of the dependence of ENDOR spectra on the static laboratory magnetic field. The dipolar hfc components yielded estimates of the electron-proton separations for each class of protons of the ethyl propenoyl moiety. Torsion angle search calculations were carried out to determine the conformational space compatible with hard-sphere nonbonded constraints and with the ENDOR-determined distance constraints. Molecular graphics analysis revealed that the propenoyl side chain of the spin-label exhibits an extended trans conformation and that the ethyl moiety of the ester group deviates significantly from coplanarity with the carboxylate--COO--atoms. The conformation of this molecule is compared with that of an analogous compound O-[3-(2,2,5,5-tetramethylpyrrolinyl-1-oxyl)-propen-2-oyl]-L- beta- phenyllactate, which has been employed as a spectroscopic substrate probe of carboxypeptidase A (L. C. Kuo, J. M. Fukuyama, and M. W. Makinen (1983) Journal of Molecular Biology 163, 63-105). The rotamer conformation of the free spin-label ester in solution, as determined in this study, and that of the enzyme-bound spin-labeled phenyllactate are compared. Differences in rotamer structure are discussed in terms of stereoelectronic principles that govern the pathway of substrate hydrolysis catalyzed by carboxypeptidase A.

Carboxypeptidases↗
PubMed1990

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.

Cyclic N-Oxides↗
PubMed1989

Dynamical structure of carboxypeptidase A.

Structural fluctuations of the apoenzyme form of carboxypeptidase A (EC 3.4.12.2) have been evaluated on the basis of molecular dynamics. The Konnert-Hendrickson refined coordinates of 2437 non-hydrogen atoms of the 307 amino acid residues derived from the X-ray structure of the holoenzyme served as the molecular model together with 548 calculated polar hydrogen atoms and 25 buried solvent molecules. Molecular dynamics simulations were carried out at 277 K, and the averaged structural properties of the protein were evaluated for the terminal 20 picosecond portion of a 48 picosecond trajectory. The average atomic displacement from the initial X-ray structure was 2.49 A for all atoms and 1.79 A for C alpha atoms. The average root-mean-square (r.m.s.) fluctuation of all atoms was 0.67 A as compared to 0.54 A evaluated from the X-ray-defined temperature factors. Corresponding r.m.s. fluctuations for backbone atoms were 0.56 A by molecular dynamics and 0.49 A by X-ray. On the basis of these molecular dynamics studies of the isolated molecule, it is shown that amino acid residues corresponding to intermolecular contact sites of the crystalline enzyme are associated with high amplitude motion. All eight segments of alpha-helix and eight regions of beta-strand were well preserved except for unwinding of the five C-terminal residues of the alpha-helix 112-122 that form part of an intermolecular contact in the crystal. Four regions of beta-strand and one alpha-helix with residues adjacent to or in the active site constitute a core of constant secondary structure and are shown not to change in relative orientation to each other during the course of the trajectory. The absence of the zinc ion does not markedly influence the stereochemical relationships of active site residues in the dynamically averaged protein. The extent of motional fluctuations of each of the subsites of substrate recognition in the active site has been evaluated. Active site residues responsible for specificity of substrate binding or splitting of the scissile bond exhibit low simulated motion. In contrast, residues in more distal sites of substrate recognition exhibit markedly greater motional fluctuations. This differential extent of dynamical motion is related to structural requirements of substrate hydrolysis.

Amino Acid Sequence↗
PubMed1988

Correlation of polarized absorption spectroscopic and X-ray diffraction studies of crystalline cytosolic aspartate aminotransferase of pig hearts.

Absorption spectra of large, well-formed crystals of cytosolic aspartate aminotransferase have been recorded using plane polarized light. Making use of measurements of crystal thickness we have calculated extinction coefficients with the electric vector of the light parallel to both the a and c axes of the crystals of the enzyme in space group P2(1)2(1)2(1). The spectra have been resolved into components with lognormal distribution curves and the resulting integrated intensities have been used to calculate the c/a polarization ratios for the absorption bands of the bound co-enzyme pyridoxal 5'-phosphate. From the polarization ratio and the co-ordinates of the co-enzyme ring atoms, provided by X-ray crystallography, we have assigned principal molecular directions of the transition dipole moment within the plane of the co-enzyme ring. Of two possible orientations, only one predicts the correct crystal extinction coefficients for the 436 nm band. In this orientation, when viewed from the B face of the ring (i.e. looking into the active site of the enzyme), the transition moment is related to the N-1-C-4 axis of the ring by counterclockwise rotation by 27 degrees. A tentative assignment of the principal molecular directions of the transition moment has also been made for the 368 nm band of the high pH form of the enzyme. In each case, the plane of the co-enzyme ring was located from the atomic co-ordinates of the ring atoms and of those atoms attached directly to the ring. The projection of the N-1 to C-4 axis on to this plane was used to evaluate the orientation of the transition moment, which was presumed to lie precisely within the plane of the ring. We have tilted this plane systematically to evaluate the error in transition moment direction resulting from uncertainties in the atomic co-ordinates. When 2-methylaspartate is diffused into the crystals if forms a Schiff base with the co-enzyme in which the ring has tilted about 32 degrees from its original position and the polarization ratio of the 436 nm band drops from 1.6 in the free enzyme to about 0.38. On the assumption that the orientation of the transition moment within the co-enzyme does not change during this rotation, this value of the polarization ratio is within experimental error of that predicted from X-ray structures on the two forms. The 2-methylaspartate binds only to subunit 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Solvent structure and enzyme activity.

Enzymes are fluctuating particles in thermal equilibrium with their solvent environment. A variety of models of enzyme action have postulated selective excitation of enzyme vibrational modes or triggering of correlated motion of catalytic groups through collisions with solvent particles as the basis of catalytic activity. Solvent composition and structure are expected to influence such interactions. Solutes such as p-dioxane, t-butanol, and tetraalkylammonium chlorides are known to be strong perturbants of the structure of water. However, when the kinetic parameters of two enzymes, carboxypeptidase A and alpha-chymotrypsin, were examined carefully in aqueous mixtures containing these solutes, no significant influence of solvent structure or mass composition on the catalytic rate constant was found. The results indicate, furthermore, that, within the low viscosity limit, fluctuations in enzyme structure that are responsible for activated processes in the catalytically rate limiting step appear not to be significantly influenced by dynamic processes in the bulk solvent.

Binding Sites↗
PubMed1984

Structural basis and dynamics of the fiber-to-crystal transition of sickle cell hemoglobin.

The kinetics of the assembly of structurally distinct, polymeric aggregates constituting the fiber-to-crystal transition of sickle cell hemoglobin in slowly stirred, deoxygenated solutions has been studied with the use of electron microscopy as a function of pH, as a function of the crystal structures of mutant forms of human deoxyhemoglobins employed as nucleating seeds, and as a function of hemoglobin S chemically modified at the Cys F9 (beta 93) position. The temporal order of appearance of fibers of approximately 210 A diameter, bundles of aligned fibers, macrofibers of greater than or equal to 650 A diameter, and microcrystals is observed. Microscopic fragments of end-stage crystals formed under slowly stirred conditions and introduced as nucleating seeds enhance the rate of crystallization only when added prior to the formation of large bundles of aligned fibers, while microscopic seed crystals added after the formation of bundles of aligned fibers do not alter the rate of crystallization. Over the pH range 6.3 to 7.1, the presence of macrofibers does not influence modulation of the kinetics of the transition with seed crystal fragments. Microscopic seed crystals of deoxyhemoglobin S and deoxyhemoglobin C formed under acidic conditions (pH less than 6.5) have a comparable influence on the kinetics of the fiber-to-crystal transition to that of end-stage crystals. Microscopic seed crystals of deoxyhemoglobin C formed under alkaline conditions (pH greater than 6.5) enhance the formation of macrofibers but do not alter the rate of crystallization. Under conditions associated with enhanced formation of macrofibers, metastable microscopic crystals having axial periodicities of approximately 64 A and approximately 210 A are observed in the intermediate phase of the transition, while end-stage crystals have axial unit cell dimensions identical to those of deoxyhemoglobin S crystallized from polyethylene glycol solutions of pH less than 6.5. Although the metastable crystals may arise from fragments of macrofibers, it is shown that they cannot be transformed directly into end-stage crystals under slowly stirred conditions without undergoing dissolution. These results stipulate that the pathway of the fiber-to-crystal transition proceeds according to the reaction: (Formula: see text) wherein the rate-limiting step is the alignment of fibers into large bundles, and macrofibers are not an intermediate of the fiber-to-crystal transition.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization↗
PubMed1983

Heme-heme orientation and electron transfer kinetic behavior of multisite oxidation-reduction enzymes.

Analysis of the polarized single-crystal absorption spectra of cytochrome cd1 of Pseudomonas aeruginosa shows that the heme c and heme d1 groups in each subunit are oriented perpendicularly to each other in both oxidized and reduced forms of the enzyme. These results, together with those of previous kinetic studies, indicate that a perpendicular heme-heme orientation may be an important factor in specifying kinetically slow steps in a sequential series of electron transfer reactions.

Cytochrome c Group↗
PubMed1983

Neutral metal-bound water is the base catalyst in liver alcohol dehydrogenase.

The catalytic role of the active site metal-water complex in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) is investigated on the basis of a comparative analysis of the pH dependence of steady-state kinetic parameters of the native and active-site-specific Co2+-reconstituted enzyme and on the basis of assignment of the coordination environment of the Co2+ by electron paramagnetic resonance methods. The pH dependence of the kinetic parameters for the oxidation of benzyl alcohol reveals two ionizations (pK1 approximately equal to 6.7; pK2 approximately equal to 10.6) that govern kcat and belong to the ternary enzyme-NAD+-alcohol complex and two ionizations (pK1' approximately equal to 7.5; pK2' approximately equal to 8.9) that govern kcat/Km and belong to the binary enzyme-NAD+ complex. The ionizations pK2 and pK2' decrease by 0.5-1 pK alpha unit upon replacement of the active site Zn2+ by Co2+. A similar metal ion dependence of pK2 and pK2' is observed for the oxidation of 2-propanol. We attribute these ionizations to a metal-bound water molecule. The zero-field splitting energy of the Co2+ in the binary enzyme-NADH complex and the ternary enzyme-NADH-CF3CH2OH complex is approximately equal to 22 cm-1, indicative of a pentacoordinate species. Binding of a water molecule to the metal ion as the fifth ligand in the ternary enzyme-NADH-CF3CH2OH complex is confirmed on the basis of magnetic interactions of H2(17)O with Co2+. The results indicate that the active site metal ion in catalytically competent ternary enzyme-coenzyme-substrate complexes is pentacoordinate and is ligated by a neutral water molecule in the physiological pH range. We suggest that the neutral metal-bound water molecule serves as the base catalyst for proton abstraction in alcohol oxidation.

Alcohol Dehydrogenase↗
PubMed1982

Hydrolysis of esters by carboxypeptidase A requires a penta-coordinate metal ion.

The catalytic role of the metal ion in bovine carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) is investigated by application of cryoenzymologic and electron paramagnetic resonance methods with use of the Co2+-reconstituted enzyme. Incorporation of 17O into oxygen-donor ligands induces a substantial change in the spin-lattice relaxation probability of the paramagnetic ion. While a change in spin-lattice relaxation is observed for the free Co2+-enzyme in 17O-enriched water, no change is observed for the enzyme complexed to glycyl-L-tyrosine. These results are consistent with x-ray crystallographic studies showing that the metal-bound water molecule in the active site is displaced upon binding of the peptide inhibitor. A change in spin-lattice relaxation of the Co2+ ion in the mixed anhydride, acyl-enzyme intermediate formed with the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate is observed when 17O is enriched either into water or into the carbonyl oxygen position of the scissile bond of the substrate. Since the protein supplies three amino acid side chains as ligands to the metal ion, these results indicate that the metal ion is altered from a tetra-coordinate species in the free enzyme to a penta-coordinate species in the acyl-enzyme reaction intermediate. In addition, the results provide structural support for our assignment of ionization of a metal-bound water molecule in rate-limiting deacylation (Makinen, M. W., Kuo, L. C., Dymowski, J. J., and Jaffer, S. (1979) J. Biol. Chem. 254, 356-366) and affirm that the metal-hydroxide species is the nucleophile responsible for the breakdown of the mixed anhydride reaction intermediate of carboxypeptidase A.

Animals↗
PubMed1981

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (EPR) methods with use of the active-site-specific Co2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme--NAD+ complex shows approximate axial symmetry of the metal ion site, indicating that binding of the coenzyme induces a structural alteration in the active-site region. This environment is not significantly altered further by binding of the competitive inhibitor pyrazole. To assign the coordination number of the active-site metal ion, the zero-field splitting was determined on the basis of the temperature dependence of the spin--lattice relaxation of the Co2+ ion. The zero-field splitting energies are approximately 9 cm-1 for the free Co2+-reconstituted enzyme and approximately 46 and approximately 47 cm-1 for the enzyme--NAD+ and enzyme--NAD+--pyrazole complex, respectively. On the basis of studies of structurally defined small molecule complexes, these values are compatible with a tetracoordinate metal ion in the active site of the free enzyme but a pentacoordinate metal ion in the binary enzyme--NAD+ complex and in the ternary enzyme--NAD+--inhibitor complex and, therefore, presumably also in the catalytically active ternary enzyme--NAD+--alcohol complex formed in the course of alcohol oxidation.

Alcohol Dehydrogenase↗
PubMed1980

Electron microscope study of the kinetics of the fiber-to-crystal transition of sickle cell hemoglobin.

The intermediates and the rate-limiting step in the crystallization of deoxygenated sickle hemoglobin have been determined by a kinetic study with the use of electron microscopy. In slowly stirred solutions of deoxygenated hemoglobin S [Pumphrey, J. & Steinhardt, J. (1977) J. Mol. Biol. 112, 359--375], the sequential appearance of fibers have a diameter of approximately equal to 210 A, bundles of aligned fibers in well-ordered arrays, "thick" fibers of approximately equal to 470 A diameter, and microcrystals is observed. Only the fibers having a diameter of approximately equal to 210 A and bundles of aligned fibers are assigned as kinetically important intermediates of the fiber-to-crystal transition. Addition of microscopic seed crystals obtained from slowly stirred solutions of deoxyhemoglobin S to a solution composed of only fibers and hemoglobin monomers results in more rapid crystallization than in control solutions. Addition of seed crystals after the formation of bindles of aligned fibers does not alter the overall kinetics of crystallization. The results demonstrate that alignment of fibers is the rate-limiting step in the crystallization process and results in formation of nucleation sites for crystal growth.

Anemia, Sickle Cell↗
PubMed1979

Synthesis of chromophoric, spin label enzyme substrates useful for cryoenzymology.

The spin label nitroxide derivative 3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oic acid has been synthesized and characterized by chemical methods. It is a useful intermediate in the preparation of a new class of chromophoric spin label substrates for enzyme studies, as shown by the synthesis of O-3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oyl-L-beta-phenyllactic acid, a specific ester substrate of bovine pancreatic carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2). Kinetic parameters of the esterolytic reaction are conveniently determined by UV spectrophotometric methods, and a reaction intermediate can be stabilized in fluid cryosolvent mixtures at subzero temperatures. Results are presented of preliminary electron spin resonance studies to demonstrate that structural relationships of the spin label substrate in a catalytically active configuration to active site residues can be determined for this low temperature-stabilized reaction intermediate. This substrate thus demonstrates the utility of this new class of spin label derivatives for characterization of enzyme reaction intermediates stabilized by cryoenzymologic techniques.

Animals↗
PubMed1979

Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.

Animals↗
PubMed1979

Structure of carboxymyoglobin in crystals and in solution.

The configuration of the heme-carbonyl group upon binding of carbon monoxide to sperm whale myoglobin (Mb) in crystals is evaluated on the basis of infrared spectroscopic methods. Multiplets of the totally symmetric C-O stretching mode are observed for the heme-bound ligand near 1933, 1944, and 1967 cm-1, corresponding to three different heme-carbonyl conformers. Variations in the relative proportions of these conformers can be induced by incorporation of small fractions of metMb or deoxyMb into MbCO crystals. The configuration of the iron-carbonyl with respect to the immediate coordination environment of the heme iron is assigned for each v(CO) stretching frequency on the basis of a detailed comparison of the three-dimensional structures of the heme environments of MbCO, metMb, and deoxyMb defined by crystallographic methods. The structures of the three heme-carbonyl conformers account for the v(CO) infrared absorption bands that can be observed for MbCO in solution.

Animals↗