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The complete ‘centre-of-subunit structure’ of the multisubunit enzyme DNA-dependent RNA polymerase was determined by triangulation of the subunit positions using the intersubunit distances calculated from scattering difference measurements and from the corresponding radii of gyration R. In addition to the centre-to-centre distances d between the core subunits α 2, β and βʹ presented in the preceding paper, the values of between initiation factor σ and α (8.4 ± 1.6 nm), β (4.4 ± 2.2 nm) and βʹ (10.7 ± 1.5 nm) were derived from R of σ (4.1 ± 0.3 nm) in situ and of the pairs α2 – σ (6.1 ± 0.4 nm), β – σ (5.6 ± 0.3 nm) and βʹ – σ (7.5 ± 0.4 nm) within the holoenzyme (α2β βʹσ). The structural parameters of the subunits within their molecular complex are accessible for neutron small-angle scattering measurements using labelling of the different subunits (deuteration), total reconstitution of isotopic hybrids, scattering length density matching of‘hydrogenated’ molecular parts and extended exposure times because of weak scattering effects. The overall shape of σ bound to core enzyme (α2ββʹ) proved to be identical (within experimental resolution) with σ in the isolated state measured recently by X-ray small-angle scattering. The refined shape of isolated σ was reduced to an ellipsoid which was orientated with respect to the core structure (α2 — β — βʹ) in a ’space-filling’ way around the position of the σ centre obtained by triangulation. The complete subunit arrangement of holoenzyme is shown in a three-dimensional model. [ABSTRACT FROM AUTHOR]

The core subunit arrangement α2-ββʹ within DNA-dependent RNA polymerase holoenzyme 2-ββʹσ from Escherichia coli was investigated by neutron small-angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C-bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits α2, β and βʹ in situ and for the determination of the intersubunit centre-to-centre distances. THe contrast between the buffer and the remaining ‘hydrogenated’ enzyme vanished if the buffer contains 42% 2H2O (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required: molecular functions, like enzyme activity, were completely preserved. The orientations of the core subunits within the holoenzyme were derived by comparing theoretical and experimental pair distance distribution functions, P(r), obtained from the scattering intensity differences of the pair-labelled (e.g. both β and βʹ labelled) and both mono-labelled molecules by direct Fourier transformations. Additional, the subunit shapes were defined by P(r) analyses. The arrangement of the stable core structure within the holoenzyme, which contains σ as a dissociable factor, is presented in a three-dimensional model [ABSTRACT FROM AUTHOR]

p-Amidinophenyl esters of an enantiomeric pair of N-(2,4-dinitrophenyl)alanine (N2Ph-Ala) were both efficiently hydrolyzed by trypsin. The acylation and deacylation rate constants for the D-isomer are 1/2.5 of those for the L-isomer. Slow rates of deacylation of the two substrates made is possible to prepare the pair of enantiomeric acyl-trypsins. Circular dichroic (CD) spectra of the purified acyl-typsins revealed that the two extrinsic chromophores are somewhat differently oriented in the chiral environment of the active site, although both chromophores could couple intermolecularly with similar intrinsic chromophores near the active site. When p-amidinophenol was added, not only was the deacylation rate of N2Ph-DAla-trypsin noticeably increased, but also the transient CD sprectrum of the enzyme derivative changed markedly in comparison with that of the L-derivative. The observations indicate that the two enantiomeric acyl groups at the active site are situated in different microenvironments. [ABSTRACT FROM AUTHOR]