The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase which we are using as a model for cold-active enzymes. Divergent amino acid residues in this 78 kDaprotein were identified through comparative genomics and hypothesized to be important for cold-activity. Six amino acid residues were previously mutated and five were shown by steady-state kinetic analysis to have altered temperature-dependent catalytic activity profiles via effects on Km and/or kcat compared to the wild-type enzyme. In this follow-up study, double-mutated enzymes were constructed and tested for temperature effects, including two new tandem residue pairs (N180T/A181T and T383A/S384A), and pairwise combination of the single residue mutations (N251D, F387L, I476V, V482L). All double-mutated enzymes were found to be more catalytically active at moderate and/or less active at colder temperatures than wild-type, with both Km and kcat effects observed for the two tandem mutations. For pairwise combinations, a Km effect was seen when the surface exposed F387L mutation located in a domain A TIM barrel alpha helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface exposed mutation I476V located in a coiled region of domain B 25 Å from the active site was paired with N251D or V482L, a kcat effect was observed. These results indicate that temperature-dependent kinetic effects may be complex and subtle and are mediated by a combination of a small number of residues distant from the active site via changes to the hydration shell and/or perturbation of internal packing.

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