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S H Koenig

Publications and source records attributed to S H Koenig.

At least 109 records · Page 6Linked to original sources
PubMed1978

Conformation as the determinant of saccharide binding in concanavalin A: Ca2+-concanavalin A complexes.

The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.

Apoproteins↗
PubMed1978

Magnetic cross-relaxation among protons in protein solutions.

The magnetic spin-lattice relaxation rates of solvent water nuclei are known to increase upon addition of diamagnetic solute protein. This enhancement of the relaxation rate is a function of magnetic field, and the orientational relaxation time of the protein molecules can be deduced from analysis of the field-dependent relaxation rates. Although the nature of the interactions that convey information about the dynamics of protein motion to the solvent molecules is not established, it is known that there is a contribution to the relaxation rates of solvent protons that plays no role in the relaxation of solvent deuterons and 17O nuclei. We show here that the additional interaction arises from a cross-relaxation process between solvent and solute protons. We introduce a heuristic three-parameter model in which protein protons and solvent protons are considered as two separate thermodynamic systems that interact across the protein-solvent interface. The three parameters are the intrinsic relaxation rates of each system and a cross-relaxation term. The sign of the latter term must always be positive, for all values of magnetic field, in order for magnetization energy to flow from the hotter to the cooler system. We find that the magnetic field-dependence of the cross-relaxation contribution is much like that of the remaining solvent proton relaxation, i.e., about the same as the deuteron relaxation field dependence. This finding is not compatible with the predictions of expressions for the cross-relaxation that have been used by other authors, but not applied to data over a wide range of magnetic field strength. The model predicts that the relaxation behavior of both the protein protons and the solvent protons is the sum of two exponentials, the relative contributions of which would vary with protein concentration and solvent isotopic composition in a fashion suggestive of the presence of two classes of protein protons, when there is in reality only one. This finding has immediate implications for the interpretation of published proton relaxation rates in complex systems such as tissues; these data should be reexamined with cross-relaxation taken into account.

Alkaline Phosphatase↗
PubMed1976

Protein rotational relaxation as studied by solvent 1H and 2H magnetic relaxation.

Earlier studies of the magnetic field dependence of the nuclear spin magnetic relaxation rate of solvent protons in solutions of diamagnetic proteins have indicated that this dependence (called relaxation dispersion) is related to the rotational Brownian motion of solute proteins. In essence, the dispersion is such that 1/T1 (the proton spin-lattice relaxation rate) decreases monotonically as the magnetic field is increased from a very low value (approximately 10 Oe); the dispersion has a point of inflection at a value of magnetic field which depends on protein size, shape, concentration, temperature, and solvent composition. The value of the proton Larmor precession frequency nu(c) at the inflection field appears to relate to tau (R), the rotational relaxation time of the protein molecules. We have measured proton relaxation dispersions for solutions of various proteins that span a three-decade range of molecular weights, and for one sample of transfer ribonucleic acid. We have also measured deuteron relaxation dispersions for solutions of three proteins: lysozyme, carbonmonoxyhemoglobin, and Helix pomatia hemocyanin with molecular weight 900 000. A quantitative relationship between both proton and deuteron dispersion data and protein rotational relaxation is confirmed, and the point is made that magnetic dispersion measurements are of very general applicability for measuring the rotational relaxation rate of macromolecules in solution. It has been previously shown that the influence of proton motion on the relaxation behavior of the solvent is not due to exchange of solvent molecules between the bulk solvent and a hydration region of the protein. In the present paper, we suggest that the interaction results from a long range hydrodynamic effect fundamental to the situation of large Brownian particles in an essentially continuum fluid. The general features of the proposed mechanism are indicated, but no theoretical computations are presented.

Deuterium↗
PubMed1976

Intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and cell-free solutions.

We have measured the intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and in cell-free solutions, and have compared the results with similar data for liganded normal adult hemoglobin. The experiments involve the measurement of the spin-lattice relaxation time T1 of protons of solvent water molecules, as a function of an externally applied static magnetic field. From such data, one can derive a correlation time tauc, for each sample, which is a measure of the time taken for a hemoglobin molecule to randomize its orientation due to Brownian motion. Thus tauc is a measure of the freedom of rotational motion, on a molecular or microscopic level, of hemoglobin molecules. Intermolecular interactions will reduce this freedom of motion and lengthen tauc. We find that oxygenated sickle hemoglobin molecules have an additional intermolecular interaction not found for normal hemoglobin. This extra interaction is increased by the presence of either inorganic phosphate or diphosphoglycerate, and is greater for sickle hemoglobin within cells than in cell-free solutions. By comparing the present results with published data on the viscosity of oxygenated sickle and normal hemoglobin, we conclude that, at concentrations comparable to intracellular values, oxygenated sickle hemoglobin molecules form aggregates several tetramers in size. The possibility exists that these aggregates are the earliest stage of fiber formation itself, the physical basis of the sickling phenomena.

Binding Sites↗
PubMed1975

Protein-water interaction studied by solvent 1H, 2H, and 17O magnetic relaxation.

Previous studies of the magnetic field dependence of the magnetic relaxation rate of solvent protons in protein solutions have indicated that this dependence (called relaxation dispersion) is related to the rotational Brownian motion of the solute proteins. In particular, the dispersion of the longitudinal (spin-lattice) relaxation rate 1/T1 shows a monotonic decrease with increasing field, with an inflection point corresponding to a proton Larmor frequency which is inversely proportional to the orientational relaxation time of the protein. We have now compared the relaxation dispersion of solvent 1H, 2H, and 17O In aqueous solutions of lysozyme (molecular weight 14,700) and 1H and 2H in solutions of hemocyanin (molecular weight 14,7 00) and 1H and 2H in solutions of hemocyanin (molecular weight 9 x 10(6)). The main experimental observation is that the dispersion of the relaxation rates of the three solvent nuclei in lysozyme solutions, normalized to their respective rates in pure water, is essentially the same. This is also true for 1H and 2H relaxation in hemocyanin solutions. These results confirm that entire solvent water molecules, rather than exchanging protons, are involved in the interaction. We have been unable to deduce the correct mechanism to explain the data, but we can eliminate several interaction mechanisms from consideration. For example, all observations combined cannot be explained by a simple two-site model of exchange, in which water molecules are either in sites on the protein with a relaxation rate characteristic of these sites, or else in the bulk solvent (the observed relaxation rate being the weighted average of the two). Also eliminated is the class of models in which the protein molecules induce a preferential partial alignment of neighboring solvent molecules, for example by electrostatic interaction of the electric dipole moments of the water with the electric fields produced by surface charges of the protein molecules. In addition, the idea that relaxation of solvent nuclei is due, in the main, to interactions with protein protons is precluded. Rather, it appears that the protein molecules influence the dynamics of the motion of solvent water molecules in their neighborhood in a manner that imposes on all the solvent molecules a correlation time for their orientational relaxation which equals that of the solute proteins.

Animals↗
PubMed1973

Solvent proton magnetic relaxation dispersion in solutions of concanavalin A.

Concanavalin A, a protein isolated from jack beans, exhibits several important biological properties, all of which are related to its ability to bind and precipitate specific polysaccharides. Concanavalin A is a dimer at pH 5.6, and has one transition-metal and one calcium-ion binding site per monomer unit of molecular weight 27,000. Both metal-ion sites must be occupied for the protein to be active. It is of interest to determine the role of the transition metal ion in Concanavalin A and its relationship to the sugar binding activity of the protein. We report the magnetic field and temperature dependences of the spin-lattice magnetic relaxation rates of solvent protons in aqueous solutions of zinc and manganese derivatives of Concanavalin A, and the influence of monosaccharide binding on these rates. The results of a leastsquares fit of the data to the theory, with five adjustable parameters, indicate that there is one rapidly exchanging water molecule ligand on the Mn(2+) ion, with a residence lifetime of 2.5 musec at 25 degrees , and with its protons 0.27 nm (2.7 A) from the Mn(2+) ion. We find that at low magnetic fields (proton Larmor frequencies below about 10 MHz), the correlation time for the dipolar interaction between the Mn(2+) electronic spin moment and the protons on the water ligand is the spin-lattice relaxation time tau(S) of the Mn(2+) moment, but that at higher magnetic fields the correlation time for the dipolar interaction is determined by the Brownian rotational tumbling of the protein, because of the substantial variation of tau(S) with magnetic field. Monosaccharide binding to manganese Concanavalin A has little effect on the relaxation rates of solvent protons, a result that indicates that the sugars do not bind directly to the transition metal in the protein.

Concanavalin A↗
PubMed1972

H 2 CO 3 as substrate for carbonic anhydrase in the dehydration of HCO 3 .

Carbonic anhydrase, a metalloenzyme containing one zinc atom per protein molecule of molecular weight 30,000, catalyzes the interconversion of CO(2) and HCO(3) (-) in solution. The rate of catalysis, among the fastest known, is pH-dependent, with a pK(Enz) near neutral. Arguments are presented to show that: (i) only the high-pH form of the enzyme is active both for the hydration and dehydration reactions (ii) at high pH there is an H(2)O ligand on the metal (not an OH(-) as is often argued), and (iii) the substrate for the dehydration reaction is the neutral H(2)CO(3) molecule. The arguments are based on data in the literature on the nuclear relaxation rates of Cl(-) ions and water protons in solutions of carbonic anhydrase, on strict application of the principle of microscopic reversibility, and on kinetic considerations. It has been argued that H(2)CO(3) cannot be the substrate for the dehydration reaction because the observed CO(2) production rate is somewhat faster than the maximum rate at which H(2)CO(3) molecules can diffuse to the active site of the enzyme. However, current models that consider HCO(3) (-) as the substrate implicity require that protons diffuse to the enzyme at an even greater rate, well outside the limitations imposed by diffusion. We consider two mechanisms to obviate the diffusion limitation problem, and conjecture that at high substrate concentration, H(2)CO(3) reaches the active site by collision with the enzyme molecule, and subsequent surface diffusion to the active site. At lower substrate concentrations, corresponding to [HCO(3) (-)] <1 mM, generation of H(2)CO(3) molecules near the enzyme by the recombination reaction H(+) + HCO(3) (-) --> H(2)CO(3) can supply an adequate flux of substrate to the active site.

Bicarbonates↗