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An intact eight‐membered water chain in drosophilid alcohol dehydrogenases is essential for optimal enzyme activity
Author(s) -
Wuxiuer Yimingjiang,
Morgunova Ekaterina,
Cols Neus,
Popov Alexander,
Karshikoff Andrey,
Sylte Ingebrigt,
GonzàlezDuarte Roser,
Ladenstein Rudolf,
Winberg JanOlof
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2012.08675.x
Subject(s) - alcohol dehydrogenase , nad+ kinase , chemistry , enzyme , alcohol , dimer , stereochemistry , biochemistry , organic chemistry
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 ( Sl ADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X‐ray crystallography of the Sl ADH 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 ( Sl ADH wt ). As for the Sl ADH wt , steady‐state kinetic studies revealed that catalysis by the Sl ADH 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 p K 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 ( Sl ADH T114V structure determined from C2 crystals) and 3RJ9 ( Sl ADH T114V structure determined from P2 1 crystals) Structured digital abstract• SlADH T114V and SlADH T114V bind by x‐ray crystallography (View Interaction: 1 , 2 )