Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 1. Brønsted parameters for cleavage of alkyl beta-D-galactopyranosides.
Seven substituted alkyl beta-D-galactopyranosides 1-OR have been prepared and shown to be fair to excellent substrates for hydrolysis catalyzed by beta-galactosidase (Escherichia coli, lac Z). Brønsted parameters of (beta 1g)k3 = -0.49 +/- 0.13 and (beta 1g)kcat/Km = -0.75 +/- 0.14, respectively, were determined at pH 8.6 for k3 (s-1), the first-order rate constant for cleavage of enzyme-bound 1-OR, and kcat/Km (M-1 s-1), the second-order rate constant for reaction of the free enzyme and 1-OR. There is a weak correlation between log Km and the pKa of the alkyl alcohol leaving group, which is attributed to stabilization of the Michaelis complex by hydrophobic interactions between the enzyme and electron-withdrawing halogen substituents at the alkoxy leaving group. These binding interactions are probably both productive and expressed in the value of kcat/Km and nonproductive and expressed in the value of k3. The negative values of beta 1g are inconsistent with enzymatic catalysis of endocyclic cleavage of the glycosidic bond. The values of beta 1g for enzyme-catalyzed cleavage of alkyl beta-D-galactopyranosides lie between those observed for the spontaneous (beta 1g approximately -1.25) and specific-acid-catalyzed (beta 1g approximately 0) cleavage of acetals, and these pathways are therefore excluded for the enzyme-catalyzed reaction. Removal of the metal cofactor Mg2+ from the enzyme causes a approximately 0.2 unit decrease in (beta 1g)k3 for beta-galactosidase-catalyzed cleavage of 1-OR. The interpretation of this change in beta 1g is unclear. The Brønsted coefficients for the beta-galactosidase-catalyzed reaction are consistent with participation by an essential catalytic residue in concerted general-acid catalysis of cleavage of the glycosidic bond of 1-OR and/or stabilization of developing negative charge at the alkoxy oxygen by interaction with the magnesium ion cofactor.
Richard
J P
JP
Department of Chemistry, University at Buffalo, SUNY 14260-3000, USA.
Westerfeld
J G
JG
Lin
S
S
D001665:Binding Sites; D002240:Carbohydrate Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D005697:Galactosides; D001616:beta-Galactosidase; D007785:Lactose