[Functional groups in the alpha-galactosidase active site in Cladosporium cladosporioides].
The activity of alpha-galactosidase isolated from culture fluid of micromycete Cladosporium cladosporioides (Fres.) de Vries 16,038 has been studied as affected by cations, anions and specific chemical reagents (p-chlormercurybenzoate (p-ChMB), iodacetamide, N-ethylmaleimide, L-cysteine, dithiotreitol, beta-mercaptoethanol, EDTA, o-phenanthroline, sodium azide). It has been established that Ag+ ions inhibited competitively alpha-galactosidase at pH 4.0 and 6.0, the inhibition constants (Ki) made 3.6 x 10(-5) M and 4.3 x 10(-6) M, respectively. Galactose in concentration of 1 mM to 5 mM preserved the enzyme from the negative effect of Ag+ ions, while L-cysteine did not manifest the protective effect. Ions of Hg2+ p-ChMB inhibited noncompetitively the activity of alpha-galactosidase, Ki for Hg2+ and p-ChMB made 5.7 x 10(-7) M and 4.7 x 10(-6) M, respectively. Preincubation with galactose does not preserve alpha-galactosidase from the inhibiting effect of Ag+ and p-ChMB, but th[not readable: see text] compounds (L-cysteine, dithiotreitol, beta-mercaptoethanol) restore the enzyme activity. Participation of histidine imidazole group in the catalytic action is supposed on the basis of the inhibitory and kinetic analysis. Sulphydryl groups do not take part in the catalysis but play an important role in supporting the active conformation of the protein molecule. The groups containing the atoms of metals are absent in the alpha-galactosidase molecule.
Malanchuk
V M
VM
Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, 154 Zabolotny St., Kyiv, 03143, Ukraine.
Buglova
T T
TT
Varbanets
L D
LD
Zakharova
I Ia
IIa
D001665:Binding Sites; D002384:Catalysis; D002963:Cladosporium / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D000519:alpha-Galactosidase