Author(s): R M Adams, B F Schmidt, S D Power, D A Estell, A L Palombella, K Moon, S E Mainzer, S Yoast
Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobacillus delbrückii subsp. bulgaricus was used to generate cold-sensitive variants. Two variants, P429S and L317F, were characterized kinetically in order to determine the enzymatic consequences of these mutations. The kinetic parameters Km and Vmax on the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside have been determined over a temperature range of 11-45 degrees C. Only the Vmax of the two variants was significantly different than the wild-type enzyme over the temperature range studied. The Vmax of the L317F variant is reduced proportionately at all temperatures compared to the wild-type enzyme while the value of Vmax for the P429S mutant deviates from wild-type only at lower temperatures (in 2 mM Mg2+). This temperature-dependent effect on the Vmax of P429S can be suppressed by increasing the Mg2+ concentration. The results suggest that the binding of this essential metal ion is altered in the P429S variant such that its dissociation is increased by lowering the temperature.