1. Free thiol groups were shown to be essential for tropomyosin to effect maximum inhibition of the Ca(2+)-stimulated ATPase (adenosine triphosphatase) of desensitized actomyosin but not for its activity in the regulatory-protein system. 2. The activity of tropomyosin on the Mg(2+)-stimulated ATPase in the regulatory-protein system was more susceptible to enzymic digestion and thermal denaturation than its effect on the Ca(2+)-stimulated ATPase of actomyosin. 3. Rabbit skeletal tropomyosin migrated as two distinct electrophoretic components in the presence of sodium dodecyl sulphate and urea and as four components on isoelectric focusing in urea. 4. The two main subunits present in rabbit skeletal tropomyosin, which have been named the alpha- and beta-chains, were separated by chromatography on CM-cellulose in urea at pH4.0. They were shown to be virtually identical in amino acid composition, except for their cysteine contents. The alpha(2) and beta(2) forms of tropomyosin possessed all the biological activities characteristic of normal tropomyosin preparations. 5. In skeletal muscle the alpha and beta components of tropomyosin were present in the proportion of 4:1. Somewhat lower ratios were obtained in skeletal muscle of sheep, pig and cow. 6. Tropomyosin isolated from cardiac muscle and Pecten maximus adductor muscle migrated as one band only. These tropomyosins possessed similar biological activities to those isolated from skeletal muscle.

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The ubiquitous enzyme Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) fixes atmospheric carbon dioxide within the Calvin-Benson cycle that is utilized by most photosynthetic organisms. Despite this central role, RuBisCO's efficiency surprisingly struggles, with both a very slow turnover rate to products and also impaired substrate specificity, features that have long been an enigma as it would be assumed that its efficiency was under strong evolutionary pressure. RuBisCO's substrate specificity is compromised as it catalyzes a side-fixation reaction with atmospheric oxygen; empirical kinetic results show a trend to tradeoff between relative specificity and low catalytic turnover rate. Although the dominant hypothesis has been that the active-site chemistry constrains the enzyme's evolution, a more recent study on RuBisCO stability and adaptability has implicated competing selection pressures. Elucidating these constraints is crucial for directing future research on improving photosynthesis, as the current literature casts doubt on the potential effectiveness of site-directed mutagenesis to improve RuBisCO's efficiency. Here we use regression analysis to quantify the relationships between kinetic parameters obtained from empirical data sets spanning a wide evolutionary range of RuBisCOs. Most significantly we found that the rate constant for dissociation of CO2 from the enzyme complex was much higher than previous estimates and comparable with the corresponding catalytic rate constant. Observed trends between relative specificity and turnover rate can be expressed as the product of negative and positive correlation factors. This provides an explanation in simple kinetic terms of both the natural variation of relative specificity as well as that obtained by reported site-directed mutagenesis results. We demonstrate that the kinetic behaviour shows a lesser rather than more constrained RuBisCO, consistent with growing empirical evidence of higher variability in relative specificity. In summary our analysis supports an explanation for the origin of the tradeoff between specificity and turnover as due to competition between protein stability and activity, rather than constraints between rate constants imposed by the underlying chemistry. Our analysis suggests that simultaneous improvement in both specificity and turnover rate of RuBisCO is possible.

The three-dimensional structure of tropomyosin filaments has been determined by X-ray crystallography. The ends of the molecules were located by reference to the single pair of cysteine residues. Departures from the alpha-helical-coiled coil conformation may occur in localised domains along the molecule as well as at the overlapping ends.

1. On electrophoresis in dissociating conditions the tropomyosins isolated from skeletal muscles of mammalian, avian and amphibian species migrated as two components. These were comparable with the alpha and beta subunits of tropomyosin present in rabbit skeletal muscle. 2. The alpha and beta components of all skeletal-muscle tropomyosins contained 1 and 2 residues of cysteine per 34000g respectively. 3. The ratio of the amounts of alpha and beta subunit present in skeletal muscle tropomyosins was characteristic for the muscle type. Muscle consisting of slow red fibres contained a greater proportion of beta-tropomyosin than muscles consisting predominantly of white fast fibres. 4. Mammalian and avian cardiac muscle tropomyosins consisted of alpha-tropomyosin only. 5. Mammalian and avian smooth-muscle tropomyosins differed both chemically and immunologically from striated-muscle tropomyosins. 6. Antibody raised against rabbit skeletal alpha-tropomyosin was species non-specific, reacting with all other striated muscle alpha-tropomyosin subunits tested. 7. Antibody raised against rabbit skeletal beta-tropomyosin subunit was species-specific.

Despite much experimental and computational study, key aspects of the mechanism of reduction of dihydrofolate (DHF) by dihydrofolate reductase (DHFR) remain unresolved, while the secondary DHFR-catalyzed reduction of folate has been little studied. Major differences between proposed DHF mechanisms are whether the carboxylate group of the conserved active-site Asp or Glu residue is protonated or ionized during the reaction, and whether there is direct protonation of N5 or a proton shuttle from an initially protonated carboxylate group via O4. We have addressed these questions for both reduction steps with a comprehensive set of ab initio quantum chemical calculations on active-site fragment complexes, including the carboxyl side chain and, progressively, all other polar active-site residue groups including conserved water molecules. Addition of two protons in two steps was considered. The polarization effects of the remainder of the enzyme system were approximated by a dielectric continuum self-consistent reaction field (SCRF) model using an effective dielectric constant (epsilon) of 2. Optimized geometries were calculated using the density functional (B3LYP) method and Onsager SCRF model with the 6-31G basis. Single-point energy calculations were then carried out at the B3LYP/6-311+G level with either the Onsager or dielectric polarizable continuum model. Additional checking calculations at MP2 and HF levels, or with other basis sets or values of epsilon, were also done. From the results, the conserved water molecule, corresponding to W206 in the E. coli DHFR complexes, that is H-bonded to both the OD2 oxygen atom of the carboxyl (Asp) side chain and O4 of the pterin/dihydropterin ring, appears critically important and may determine the protonation site for the enzyme-bound substrates. In the absence of W206, the most stable monoprotonated species are the neutral-pair 4-enol forms of substrates with the carboxyl group OD2 oxygen protonated and H-bonded to N3. If W206 is included, then the most stable forms are still the neutral-pair complexes but now for the N3-H keto forms with the protonated OD2 atom H-bonding with W206. A second proton addition to these complexes gives protonations at N8 (folate) or N5 (DHF). Calculated H-bond distances correlate well with those for the conserved W206 observed in many X-ray structures. For all structures with occluded M20 loop conformations (closed active site), OD2-N3 distances are less than OD2-NA2 distances, which is consistent with those calculated for protonated OD2 complexes. Thus, the results (B3LYP; epsilon = 2 calculations) support a mechanism for both folate and DHF reduction in which the OD2 carboxyl oxygen is first protonated, followed by a direct protonation at N8 (folate) and N5 (DHF) to obtain the active cation complexes, i.e., doubly protonated. The results do not support a proposed protonated carboxyl with DHF in the enol form for the Michaelis complex, nor an ionized carboxyl with protonated enol-DHF as a catalytic intermediate. However, as additional calcula...

A coupled quantum mechanical and molecular mechanical (QM/MM) model based on the AM1, MNDO, and PM3 semiempirical molecular orbital methods and the TIP3P molecular mechanics model for liquid water is presented. The model was parameterized for each of the three molecular orbital methods using the aqueous solvation free energies of a wide range of neutral organic molecules, many of which are representative of amino acid side chains. The fit to the experimental solvation free energies was achieved by varying the radii in the van der Waals (vdW) terms for interactions between the solute, which was treated quantum mechanically, and the molecular mechanics (TIP3P) solvent molecules. It is assumed that the total free energy can be obtained as the sum of components derived from the electrostatic terms in the Hamiltonian plus a generally smaller “nonelectrostatic” term. The electrostatic contributions to the solvation free energies were computed using molecular dynamics (MD) simulation and thermodynamic integration techniques; the nonelectrostatic contributions were taken from the literature. It was found that the experimental free energies could be reproduced accurately (to within 1 kcal/mol) from the MD simulations, provided that the vdW parameter associated with hydrogen bonding (H bonding) was allowed to have different values depending on the QM method (AM1, MNDO, or PM3) and the type of functional group involved in the H bonding. Moreover, the radial distribution functions obtained from the MD simulations using such a parameterization scheme showed the expected H‐bonded structures between the solute and molecules of the solvent. The solvent‐induced dipole moments also compared favorably with the results of other QM/MM model calculations. © 1997 John Wiley & Sons, Inc. J Comput Chem 18: 1496–1512, 1997