Comparison of several new chromogenic galactosides as substrates for various beta-D-galactosidases.
The kinetic characteristics of beta-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli beta-D-galactosidase resulting in low Km values. The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed to interactions not involving the catalytic site. When the product of the maximum observed velocity (Vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed. The beta-D-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.
Pócsi
I
I
Institute of Biochemistry, L. Kossuth University, Debrecen, Hungary.
Taylor
S A
SA
Richardson
A C
AC
Smith
B V
BV
Price
R G
RG
D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002417:Cattle; D002863:Chromogenic Compounds / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology; D001616:beta-Galactosidase / Q000378:metabolism*
D002863:Chromogenic Compounds; D005697:Galactosides; D001616:beta-Galactosidase