ACTIVATION OF HORSE LIVER ALCOHOL-DEHYDROGENASE UPON SUBSTITUTION OF TRYPTOPHAN-314 AT THE DIMER INTERFACE

Horse liver alcohol dehydrogenase contains two tryptophan residues per subunit, Trp-15 on the surface of the catalytic domain and Trp-314 bu ried in the interface between the subunits of the dimer, We studied th e contributions of the tryptophans to fluorescence and catalytic dynam ics by substituting Trp-314 with a leucine residue and making two comp ensatory mutations that were required to obtain a stable protein, lead ing to the triple mutant M303F-L308I-W314L enzyme, The substitutions i ncreased by two- to sixfold the turnover numbers for ethanol oxidation , acetaldehyde reduction, and the dissociation constants of the coenzy mes. The rate of the exponential burst phase for the transient oxidati on of ethanol increased slightly, but the rate of dissociation of the enzyme-NADH complex still limited turnover of ethanol, as for wild-typ e enzyme, The three substitutions at the dimer interface apparently ac tivate the enzyme by allowing more rapid conformational changes that a ccompany coenzyme binding, probably due to movement of the loop contai ning residues 293 to 298, The emission spectrum of M303F-L308I-W314L e nzyme, which contains Trp-15, was redshifted compared to wild-type enz yme. Time-resolved fluorescence measurements with the triple mutant sh ow that the decay of Trp-15 is dominated by a similar to 7-ns componen t. In the mutant enzyme with Trp-15 substituted with phenylalanine, th e decay of Trp-314 is dominated by a similar to 4-ns component. Solute quenching data for wild-type enzyme and the mutants show that only Tr p-15 is exposed to iodide and acrylamide, whereas Trp-314 is inaccessi ble, The luminescence properties of the tryptophan residues in the mut ated enzymes are consistent with conclusions from studies of the wild- type enzyme [M. R. Eftink, 1992, Adv. Biophys. Chem. 2, 81-114]. (C) 1 998 Academic Press.