Premium
pH‐rate profiles support a general base mechanism for galactokinase ( Lactococcus lactis )
Author(s) -
Reinhardt Laurie A.,
Thoden James B.,
Peters Greg S.,
Holden Hazel M.,
Cleland W.W.
Publication year - 2013
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2013.07.017
Subject(s) - lactococcus lactis , deprotonation , chemistry , galactokinase , galactose , enzyme , mutant , biochemistry , stereochemistry , lactic acid , bacteria , biology , organic chemistry , escherichia coli , ion , genetics , gene
Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α‐ d ‐galactose to galactose‐1‐phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis . Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (<10 000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C‐1 hydroxyl group of galactose. The pH‐ k cat profile of the forward reaction has a p K a of 6.9 ± 0.2 that likely is due to Asp183. The pH‐ k cat / K Gal profile of the reverse reaction further substantiates this role as it is lacking a key p K a required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100‐fold lower activity than that for the wild‐type enzyme, thus suggesting that Arg36 lowers the p K a of the C‐1 hydroxyl to facilitate deprotonation.