Characterization of two cold-sensitive mutants of the beta-galactosidase from Lactobacillus delbruckii subsp. bulgaricus

Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobacillus delbruckii 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 parame...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-02, Vol.269 (8), p.5666-5672
Main Authors: Adams, R.M, Yoast, S, Mainzer, S.E, Moon, K, Palombella, A.L, Estell, D.A, Power, S.D, Schmidt, B.F
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Analytical, structural and metabolic biochemistry
BETA GALACTOSIDASA
BETA GALACTOSIDASE
beta-Galactosidase - chemistry
beta-Galactosidase - genetics
beta-Galactosidase - metabolism
Biological and medical sciences
CATION
CATIONES
Cold Temperature
Enzymes and enzyme inhibitors
FRIO
FROID
Fundamental and applied biological sciences. Psychology
HIDROLISIS
Hydrolases
HYDROLYSE
Hydrolysis
INHIBICION
INHIBITION
Kinetics
Lactobacillus - enzymology
LACTOBACILLUS DELBRUECKII
LACTOSA
LACTOSE
Lactose - metabolism
MAGNESIO
MAGNESIUM
Magnesium - metabolism
METAL
METALES
Molecular Weight
MUTACION
MUTACION INDUCIDA
MUTANT
MUTANTES
MUTATION
MUTATION PROVOQUEE
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Summary:Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobacillus delbruckii 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
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)37512-9