Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 4. Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermediates of E461G and E461Q beta-galactosidases.
Second-order rate constants for transfer of the beta-D-galactopyranosyl group from the galactosyl-enzyme intermediates of the galactosyl transfer reactions catalyzed by E461G and E461Q beta-galactosidases to anionic nucleophiles have been determined. The second-order rate constant for reaction of the galactosylated E461G enzyme with azide ion is 4900 M-1 s-1. By contrast, there is no detectable reaction of the galactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order rate constant kcat/Km for catalysis of cleavage of beta-D-galactopyranosyl azide, which is the microscopic reverse of the reaction of azide ion with the galactosyl-enzyme intermediate. These data show that the E461G mutation causes a more than 8000-fold increase in the equilibrium constant for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to azide ion. We propose that this change represents the requirement for the coupling of galactosyl transfer from the native enzyme to the thermodynamically unfavorable protonation of the carboxylate group of Glu-461, but the expression of the full chemical affinity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461. The reactions of acetate, butyrate and methoxyacetate ions with the galactosylated E461G enzyme and of acetate with the galactosylated E461Q enzyme give both the corresponding beta-galactopyranosyl derivatives and D-galactose, and the formation of the latter represents formal catalysis of the reaction of water with the galactosylated enzyme. However, the reaction of formate ion with the galactosylated E461G enzyme gives only D-galactose. These results suggest that carboxylate anions can take the place of the excised propionate side chain of Glu-461 to provide general base catalysis of the reaction of water with the galactosyl-enzyme intermediates. The relative reactivity of anionic nucleophiles toward the covalent galactosyl-enzyme intermediate of the reactions catalyzed by the E461G enzyme is similar to that observed for partitioning of stable carbocations in water. This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbenium ion intermediate to reaction with external nucleophilic reagents.
Richard
J P
JP
Department of Chemistry, University at Buffalo, SUNY 14260-3000, USA.
Huber
R E
RE
Heo
C
C
Amyes
T L
TL
Lin
S
S
D000085:Acetates / Q000378:metabolism / Q000494:pharmacology; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005561:Formates / Q000378:metabolism / Q000494:pharmacology; D005690:Galactose / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009598:Nitrophenylgalactosides / Q000378:metabolism; D017354:Point Mutation; D013329:Structure-Activity Relationship; D014867:Water / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000085:Acetates; D001386:Azides; D005561:Formates; D005697:Galactosides; D014867:Water; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D001616:beta-Galactosidase; D005690:Galactose