ABSTRACT
Nitration of tyrosine residues was performed on Bacillus circulans E 192 cyclomaltodextrin glucanotransferase (CGTase) using tetranitromethane (TNM). A maximum of 15 out of 28 tyrosine residues is modified with 8 mM TNM, entailing a concomitant loss of enzymic activity and tryptophan fluorescence. Spectroscopic studies suggest that these two phenomena are related to an impairment of the enzyme conformation as a consequence of the tyrosine nitration. The presence of 5 mM acarbose during the CGTase nitration results in the protection of one tyrosine residue and the rate of inactivation is reduced 9.4-fold. These results support a contribution of a tyrosine residue in the CGTase catalytic site. The nitration of CGTase also entails a decrease in the enzyme's affinity for a beta-cyclodextrin (beta-CD) co-polymer. Kinetic and analytical investigations on isolated modified enzymes support the concept that this phenomenon is unrelated to the modification of tyrosine residues, but rather concerns a side reaction of the reagent occurring at the raw-starch-binding site of the CGTase.
Subject(s)
Bacillus/enzymology , Glucosyltransferases/metabolism , Nitrates/metabolism , Tetranitromethane/metabolism , Acarbose , Circular Dichroism , Glucosyltransferases/antagonists & inhibitors , Glucosyltransferases/drug effects , Kinetics , Spectrometry, Fluorescence , Trisaccharides/pharmacology , Tyrosine/metabolismABSTRACT
The cyclomaltrodextrin glucanotransferase (CGTase) [1,4-alpha-D-glucan:4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), EC 2.4.1.19] from Bacillus circulans E 192 has been purified to homogeneity by Cetavlon treatment, ammonium sulfate precipitation, DEAE Trisacryl M chromatography, Q Fast Flow chromatography, and affinity on beta-cyclodextrin-Sepharose 4B. Two isoenzymes were separated by FPLC on a Mono Q column. Their isoelectric points were estimated as 6.7 and 6.9 and they represented 13 and 87%, respectively, of the initial activity. Their molecular weight, pH, and temperature optima were estimated as 78,000, 5.5, and 60 degrees C, respectively. Kinetic parameters indicated that both enzymes had the same properties; they preferentially modified high-molecular-weight substrates to produce cyclodextrins. The apparent Vmax and Km values for soluble starch were 43 mumol of beta-cyclodextrin/min/mg of protein and 0.57% (w/v), respectively. Although this CGTase is not markedly thermostable, it is protected against heat denaturation by substrate, product, and/or calcium ions. The ratios of alpha-, beta-, and gamma-cyclodextrins produced have been determined as 1/7/2 in the initial phase of the reaction and 3/3/1 at equilibrium.
Subject(s)
Bacillus/enzymology , Glucosyltransferases/isolation & purification , Amino Acid Sequence , Amino Acids/analysis , Electrophoresis, Polyacrylamide Gel , Glucosyltransferases/chemistry , Glucosyltransferases/metabolism , Hydrogen-Ion Concentration , Isoelectric Point , Kinetics , Molecular Sequence Data , TemperatureSubject(s)
Aldose-Ketose Isomerases , Carbohydrate Epimerases/genetics , Mutagenesis, Site-Directed , Streptomyces/enzymology , Amino Acid Sequence , Carbohydrate Epimerases/metabolism , Enzyme Stability , Escherichia coli/genetics , Hot Temperature , Molecular Sequence Data , Plasmids , Restriction Mapping , Streptomyces/geneticsABSTRACT
The complete purification of renin raises difficult problems due to its extremely low concentration in kidney (less than 1/50,000 of total proteins). The complete purification of hog kidney renin has been realized on a large scale, starting from 300 kg of fresh hog kidneys. 14.6 mg of pure renin were obtained with an overall yield of 4%. The purification procedure involved 14 steps. The enzyme was extracted at pH 3.5. Subsequent purification steps were performed in the presence of protease inhibitors to decrease renin proteolysis. These steps included an ammonium sulfate precipitation and a batch-chromatography on DEAE-cellulose. The major purification step was an affinity chromatography on Sepharose-hexamethylene-diaminopepstatin. The enzyme obtained was further purified by molecular sieving gel filtration and isoelectric focusing.
