RESUMO
Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.
Assuntos
Aspergillus/enzimologia , Poligalacturonase/metabolismo , Tirosina/fisiologia , Anidridos Acéticos/farmacologia , Acetilação , Sequência de Aminoácidos , Ativação Enzimática , Reativadores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Hidroxilamina , Hidroxilaminas/farmacologia , Imidazóis/farmacologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Poligalacturonase/química , Poligalacturonase/efeitos dos fármacos , Análise de Sequência , Tetranitrometano/farmacologia , Tirosina/análiseRESUMO
The mechanism of action of the specific D-galacturonan digalacturonohydrolase [poly-(1----4)-alpha-D-galactosiduronate digalacturonohydrolase, EC 3.2.1.82] of Selenomonas ruminantium was investigated by using reducing-end [1-3H]-labeled oligogalactosiduronates having degree of polymerization 3-5 as the substrates. The reaction products, incorporation and distribution of radioactivity in products, and the frequency of oligogalactosiduronate bond-cleavage were quantitatively estimated as functions of the substrate concentration. An alternative cleavage of tri(D-galactosiduronate) occurred during the enzyme reaction, indicating the participation of some bimolecular mechanism in addition to unimolecular hydrolysis in the action of the enzyme. Unimolecular hydrolysis takes place at low initial concentration of the substrate. The shifted termolecular enzyme-substrate complex formation and the subsequent galactosyluronic transfer is the predominant mechanism in degradation of tri(D-galactosiduronate) at high concentration. Tetra(D-galactosiduronate) and penta(D-galactosiduronate) are degraded by unimolecular hydrolysis at low, as well as high concentration of the substrate.
