Active Sites of Diacylglycerol Kinase from Escherichia coli Are Shared between Subunits

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Abstract

We show that residues from different subunits participate in forming the active site of the trimeric membrane protein diacylglycerol kinase (DGK) from Escherichia coli. Five likely active-site mutants were identified: A14Q, N72S, E76L, K94L, and D95N. All five of these mutants possessed significantly impaired catalytic function, without evidence of gross structural alterations as judged by their similar near-UV and far-UV circular dichroism spectra. We found that mixtures of either A14Q or E76L with N72S or K94L possessed much greater activity than the mutant proteins by themselves, suggesting that Ala14 and Glu76 may be on one half-site while Asn72 and Lys94 are on another half-site. Consistent with the shared site model, we also found that (1) peak activity of A14Q and N72S subunit mixtures occur at equimolar concentrations; (2) the maximum activity of the A14Q and N72S mixture was 20% of the wild-type enzyme, in good agreement with the theoretical maximum of 25%; (3) the activity of mutant subunits could not be recovered by mixing with the wild-type subunits; (4) a double mutant, A14Q/N72S, bearing mutations in both putative half-sites was found to inactivate wild-type subunits; (5) the concentration dependence of inactivation by the A14Q/N72S mutant could be well described by a shared site model for a trimeric protein. Unexpectedly, we found that the single mutant D95N behaved in a manner similar to the double mutant, A14Q/N72S, inactivating wild-type subunits. We propose that Asp95 plays a role in more than one active site.

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