THE IDENTIFICATION OF A LYSINE RESIDUE REACTIVE TO PYRIDOXAL-5-PHOSPHATE IN THE GLYCEROL DEHYDROGENASE FROM THE THERMOPHILE BACILLUS-STEAROTHERMOPHILUS

The glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is i nactivated by incubation with pyridoxal-5-phosphate (PALP). The comple x formed between the two can be trapped by reduction with sodium boroh ydride to yield a protein with an absorbance band at 325 nm and a fluo rescence emission band at 430 nm, typical of trapped pyridoxal-5-phosp hate moieties. Total loss of catalytic activity of the enzyme is assoc iated with the modification of approximately one equivalent of the rea gent; the incorporation of the reagent and the loss of activity can be prevented by the additional presence of the oxidised or reduced coenz yme. Peptides derived from the labelled protein have been sequenced an d have identified Lys-97 as the reactive residue. Site-directed mutage nesis has been used to replace Lys-97 by a His residue. This mutated e nzyme has no catalytic activity and fluorescence spectroscopy studies suggest that it is unable to bind NADH.