Proteolytic cleavage of pyridoxal kinase into two structural domains

Abstract

Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields 2 fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by chromatographic techniques and analyzed for interaction with the ATP analogue, trinitrophenyl-ATP, using fluorescence spectroscopy. The absorption and fluorescence properties of trinitrophenyl-ATP bound to the fragment of 24 kDa (emission maximum, 540 nm, emission anisotropy at 460 nm, 0.30, and fluorescence lifetime, γ = lns) are indistinguishable from those of the ATP analogue bound to the native enzyme. The fragment of 16 kDa does not bind trinitrophenyl-ATP. The results are consistent with the hypothesis that monomeric pyridoxal kinase is folded into 2 domains connected by a single polypeptide chain sensitive to proteolytic cleavage.