β-Galactosidases of Escherichia coli with substitutions for Glu-461 can be activated by nucleophiles and can form β-d-galactosyl adducts
✍ Scribed by Reuben E. Huber; Peter T. Chivers
- Publisher
- Elsevier Science
- Year
- 1993
- Tongue
- English
- Weight
- 672 KB
- Volume
- 250
- Category
- Article
- ISSN
- 0008-6215
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✦ Synopsis
Nucleophiles activated the catalytic actions of P-galactosidases with neutral or positively charged substitutions for Glu-461. Aliphatic carboxylic acids increased the rate of hydrolysis of o-nitrophenyl P-o-galactopyranoside if the pKa values of the carboxyl groups were > -3.5. Amino compounds activated if their pKa values were < -8.5. Imidazole, azide, and 2-mercaptoethanol also activated. Nucleophiles with high pKa values were able to activate the catalysis if the pH was high, and this showed that the lack of activation at pH 7.0 was because of protonation. Kinetic analysis showed that most of the nucleophiles that activated were bound to the active site, since the activation followed Michaelis-Menten type saturation kinetics. The binding seemed to be dependent upon the hydropho-bic&y; the longer the aliphatic chain, the stronger the binding. Gas-liquid chromatographic analysis showed that adducts of some type were formed during the reactions in the presence of many of the nucleophiles. Three of these adducts were purified and the nucleophiles were found P-linked to D-gahCtOSe.
This indicates that if an intermediate covalent bond is formed in the mechanism of P-galactosidase action and if the nucleophile reacts to displace it, the intermediate covalent bond must have the (Y configuration and involve a group other than Glu-461.