Effect of an E461G Mutation of β-Galactosidase (Escherichia coli, lac Z) on pL Rate Profiles and Solvent Deuterium Isotope Effects

✍ Scribed by John P. Richard; Reuben E. Huber; Deborah A. McCall

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 116 KB
• Volume: 29
• Category: Article
• ISSN: 0045-2068
• DOI: 10.1006/bioo.2001.1206

✦ Synopsis

An E461G mutation of ␤-galactosidase results in the disappearance of the high pL (L ϭ H, D) downward break in the rate profiles for k cat /K m for wild-type enzyme-catalyzed hydrolysis of 4-nitrophenyl ␤-D-galactopyranoside (Gal-OPNP) and a decrease from (k cat ) HOH /(k cat ) DOD ϭ 1.7 to (k cat ) HOH /(k cat ) DOD ϭ 1.2 in the solvent deuterium isotope effect. These observations provide evidence that the propionic acid side chain of Glu 461 is protonated at catalytically active free ␤-galactosidase and they are consistent with a role for this residue in Brønsted acid catalysis at the leaving group. The earlier observation that this same E461G mutation results in the loss of a downward break at high pH in the rate profile for k s for transfer of the ␤-Dgalactopyranosyl group from ␤-galactosidase to water cannot be simply explained by a mechanism in which the single side chain of Glu 461 functions to provide general acid catalysis in the rate limiting step for formation of the ␤-D-galactopyranosyl intermediate and general base catalysis of breakdown of this intermediate. Evidence is presented that there may be different catalytic mechanisms, with different roles for the side chain for Glu-461, for nucleophilic addition of water and of small alkyl alcohols to the ␤-D-galactopyranosyl reaction intermediate.