pH‐rate profiles support a general base mechanism for galactokinase ( Lactococcus lactis )

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.