Proteinchemical and kinetic features of gramicidin S synthetase

The amino-acid compositions of both enzymes of gramicidin S synthetase were determined. These proteins contain a high number of acidic amino-acid residues. Phenylalanine racemase, the light enzyme, was sequenced from the N-terminus until position 10. The kinetics of the thioester formation reactions were studied. The half-life times of these processes under substrate saturation conditions were found in the range between seconds and a few minutes. The valine activation at the heavy enzyme was detected as one of the rate-limiting steps of the biosynthesis of gramicidin S.     

Gramicidin S synthetase. Stability of reactive thioester intermediates and formation of 3-amino-2-piperidone

The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents. In the presence of 1 mM dithioerythritol the half-life times of these hydrolysis reactions are in the range of 1-90 h at 3 degrees C. The thioester complex of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester complex of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn) is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester binding site. All other activation reactions of gramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined.     

Identification of two binding sites of the D-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach for D-ribulose 1,5-bisphosphate and effectors of the carboxylation reaction.

Fluorimetric studies of the binding of d-ribulose 1,5-bisphosphate (RuP₂) and the effectors 6-phosphogluconate and fructose 1,6-bisphosphate to the d-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach were correlated with the functions of these sugar phosphates in the carboxylation reaction. These agents compete for two binding sites of the enzyme. At relatively low concentrations they bind to an allosteric site, where 6-phosphogluconate and fructose 1,6-bisphosphate display their stimulating effect on the fixation of CO₂. At higher concentrations these compounds inhibit the carboxylation reaction and compete with RuP₂ for the reaction center of the carboxylase. Preincubation of the enzyme with low concentrations of RuP₂ (0.1–5 μm) inhibits the activity of these effectors as well as the effector-induced fluorescence changes of the enzyme-2-p-toluidinonapthalene-6-sulfonate (TNS) complex by competition for the regulatory center which could be identified as the high affinity binding site of the enzyme for RuP₂ with a K_D = 0.6 μm. The deactivation of the carboxylase which is observed on preincubation of the enzyme with RuP₂ in the absence of bicarbonate and Mg²⁺ cannot be correlated to the binding of RuP₂ to the effector site. The deactivation process occurs in an RuP₂ concentration range similar to that for CO₂ fixation.     

An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins

Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.