[Spectroscopic study of the structure and intramolecular mobility of yeast pyruvate decarboxylase]. / Spektroskopicheskie issledovaniia struktury i vnutrimolekuliarnoi podvizhnosti drozhzhevoi piruvatdekarboksilazy.

Steady-state and time-resolved fluorimetry were used to study the properties of holo- and apopyruvate decarboxylase (EC 4.1.1.1, PDC) from Brewer's yeast after interaction with substrate (pyruvate), cofactor (thiamine diphosphate, ThDP) and Mg2+ ions. The analysis of the enzyme's intrinsic fluorescence as well as of its complex with the probe 2-(p-toluidinylnaphthalene)-6-sulphonate (TNS) revealed that ThDP was found at the polar region of the PDC active sites, inducing a decrease in the mobility of the protein's nearest surroundings. The fluorescent probe had three different sites of binding to the protein apoform, two of which being located at the catalytic site and having different rotation freedom. The study of the PDC complex with thiochrome pyrophosphate, a ThDP structural analogue, pointed to the occurrence of a non-polar region of the enzyme active site for pyruvate absorption besides the polar region. The binding of pyruvate to the protein does not depend upon the cofactor's binding. On the basis of the fluorescent studies a model of the ThDP and pyruvate arrangement at the PDC active site is suggested.

[Physico-chemical properties of thiamine kinase from Saccharomyces carlsbergensis yeasts]. / Fiziko-khimicheskie svoistva tiaminkinazy drozhzhei Saccharomyces carlsbergensis.

The amino acid composition and intrinsic fluorescence were studied in thiamine kinase (ES 2.7.6.2) of brewer's yeast. The enzyme molecule is characterized by higher concentrations of amino acids which promote alpha-helix formation of the protein globule, the amount of residues (cysteine, proline) either binding or folding polypeptide chains being considerably high. Amino acids of middle and low hydrophobicities were the most frequent among the amino acid residues with nonpolar R-groups. The value for the protein isoelectric point was 6.21. The eigen pH value and isoionic point were in good agreement with the isoelectric point value and amounted to 6.28. The fluorescence spectrum has a maximum at 328 nm, half-width at 53 nm and a quantum yield at 0.14 nm. The tryptophane residues were located in hydrophobic surroundings, unexposed to anion quenchers and almost unexposed to cation ones. The fluorescence and phosphofluorescence parameters were sensitive to the conformational changes in the molecule. At pH of 5-9 the protein conformation remained unchanged. The temperature rise above 40 degrees C resulted in a disturbance in the nativity of the globule. The elevation of the enzyme concentration from 0.05 to 1 mg/ml increased the polarization degree from 0.115 to 0.194, the quantum yield and the spectrum position remaining unchanged. The results obtained develop knowledge of the equilibrium system of oligomerous forms of thiamine kinase with different catalytic properties.