Regulation of lactose catabolism in Streptococcus mutans: purification and regulatory properties of phospho-beta-galactosidase.
Phospho-beta-galactosidase (P-beta-gal), the enzyme which catalyzes the first step in the metabolism of intracellular lactose phosphate, occurred at high specific activity in the cytoplasm in 12 of 13 strains of streptococcus mutans grown on lactose but not other carbon sources. The P-beta-gal from S. mutans SL1 was purified 13-fold using diethylaminoethyl-cellulose ion exchange and agarose A--0.5 M molecular exclusion column chromatography. The molecualr weight of the enzyme was estimated to be 40,000, and its pH optimum was 6.5 in three different buffer systems. P-beta-gal activity was inhibited by Co2+, Zn2+, and Cu2+, but other cations, ethylenediaminetetraacetic acid, orthophosphate, and fluoride had no effect upon enzyme activity. The kinetic response of P-beta-gal to a model substrate, o-nitrophenyl-beta-D-galactopyranoside-6-phosphate, obeyed Michaelis-Menten kinetics, and the Km for this substrate was 0.19 mM. In addition to being under genetic control, P-beta-gal activity was regulated by a number of biologically active metabolites. Enzyme activity was inhibited in a sigmoidal fashion by phosphoenolpyruvate. The M 0.5 V value for phosphoenolpyruvate was 2.8 mM, and the Hill coefficient (n) was 3. In addition, P-beta-gal exhibited strong inhibition by ATP, galactose-6-phosphate, and glucose-6-phosphate. In contrast to inhibition of P-beta-gal activity by phosphoenolpyruvate, the inhibition exerted by ATP, galactose-6-phosphate, and glucose-6-phosphate obeyed classical Michaelis-Menten kinetics; the Ki values for these inhibitors were 0.55, 1.6, and 4.0 mM, respectively.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D004790:Enzyme Induction; D005695:Galactosephosphates; D005696:Galactosidases / Q000378:metabolism*; D006600:Hexosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism*; D008970:Molecular Weight; D013295:Streptococcus mutans / Q000201:enzymology*; D001616:beta-Galactosidase / Q000302:isolation & purification / Q000378:metabolism*
D005695:Galactosephosphates; D006600:Hexosephosphates; D000255:Adenosine Triphosphate; D005696:Galactosidases; D001616:beta-Galactosidase; D007785:Lactose