Active sites of diacylglycerol kinase from Escherichia coli are shared between subunits

We show that residues from different subunits participate in forming the ac tive site of the trimeric membrane protein diacylglycerol kinase (DGK) from Escherichia coli. Five likely active-site mutants were identified: A14Q, N 72S, E76L, K94L, and D95N. All five of these mutants possessed significantl y impaired catalytic function, without evidence of gross structural alterat ions as judged by their similar near-UV and far-UV circular dichroism spect ra. We found that mixtures of either A14Q or E76L with N72S or K94L possess ed 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 tha t (1) peak activity of A14Q and N72S subunit mixtures occur at equimolar co ncentrations; (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 mutati ons in both putative half-sites was found to inactivate wild-type subunits; (5) the concentration dependence of inactivation by the A14Q/N72S mutant c ould be well described by a shared site model for a trimeric protein. Unexp ectedly, we found that the single mutant D95N behaved in a manner similar t o the double mutant, A14Q/N72S, inactivating wild-type subunits. We propose that Asp95 plays a role in more than one active site.