pH-rate profiles support a general base mechanism for galactokinase (Lactococcus lactis)
- Reinhardt, Laurie A.
- Thoden, James B.
- Peters, Greg S.
- Holden, Hazel M.
- Cleland, W.W.
DOIAbstract
AbstractGalactokinase (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 (<10000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH-kcat profile of the forward reaction has a pKa of 6.9±0.2 that likely is due to Asp183. The pH-kcat/KGal profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct pr...
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