Subunit communication in tetrameric class 2 human liver aldehyde dehydrogenase as the basis for half-of-the-site reactivity and the dominance of the oriental subunit in a heterotetramer

Data has been published showing that in heterotetrameric liver mitochondria l aldehyde dehydrogenase composed of the active (E487) and the inactive Ori ental-variant (K487) subunit, the Oriental Variant was dominant and caused the inactivation of the E487 subunit. The published structures of the enzym e showed that the glutamate at position 487 is salt bonded to an arginine ( 475) in a different subunit. Arg475 was mutated to a glutamine to test for its importance in causing the Oriental variant to be an enzyme with a high Km for NAD and a low specific activity. Unexpectedly, the R475Q mutant exhi bited positive cooperativity in NAD binding with a Hill coefficient of 2. I ndividual heterotetramers composed of subunits of E487 and K487 were produc ed by making changes to two residues on the surface of the enzyme and then co-expressing both cDNAs in E. coli. The E3K form had essentially 50% the a ctivity of the E-4 homotetrameric form while EK3 had essentially the same p roperties as did the homotetrameric K-4 Oriental variant. This showed that in a dimer pair composed of one K- and one E- subunit the K-subunit became dominant and caused the inactivation of its E-partner. Further, pre-steady state burst data and steady state kinetic data make it appear that there wa s one functioning active subunit in each of the dimer pairs that made up th e tetrameric enzyme. Thus, the half-of-the-site reactivity is a result of h aving one functioning and one non-functioning subunit in each dimer pair. T he actual structural basis for this is still not understood, but could be r elated to the E487-R475 inter-dimer salt bond. (C) 2001 Elsevier Science Ir eland Ltd. All lights reserved.