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3RJ5: Structure Of Alcohol Dehydrogenase From Drosophila Lebanonesis T114v Mutant Complexed With Nad+
An intact eight-membered water chain in drosophilid alcohol dehydrogenases is essential for optimal enzyme activity
FEBS J. (2012) 279 p.2940-2956
All drosophilid alcohol dehydrogenases contain an eight-member water chain connecting the active site with the solvent at the dimer interface. A similar water chain has also been shown to exist in other short-chain dehydrogenase/reductase (SDR) enzymes, including therapeutically important SDRs. The role of this water chain in the enzymatic reaction is unknown, but it has been proposed to be involved in a proton relay system. In the present study, a connecting link in the water chain was removed by mutating Thr114 to Val114 in Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X-ray crystallography of the SlADH(T114V) mutant revealed a broken water chain, the overall 3D structure of the binary enzyme-NAD(+) complex was almost identical to the wild-type enzyme (SlADH(wt) ). As for the SlADH(wt) , steady-state kinetic studies revealed that catalysis by the SlADH(T114V) mutant was consistent with a compulsory ordered reaction mechanism where the co-enzyme binds to the free enzyme. The mutation caused a reduction of the k(on) velocity for NAD(+) and its binding strength to the enzyme, as well as the rate of hydride transfer (k) in the ternary enzyme-NAD(+) -alcohol complex. Furthermore, it increased the pK(a) value of the group in the binary enzyme-NAD(+) complex that regulates the k(on) velocity of alcohol and alcohol-competitive inhibitors. Overall, the results indicate that an intact water chain is essential for optimal enzyme activity and participates in a proton relay system during catalysis. Database The X-ray crystallography data are available in under Protein Data Bank accession numbers 3RJ5 (SlADH(T114V) structure determined from C2 crystals) and 3RJ9 (SlADH(T114V) structure determined from P2(1) crystals) Structured digital abstract * SlADH T114V and SlADH T114V bind by x-ray crystallography (View Interaction: 1, 2).