Role of pyridoxal 5 '-phosphate in the structural stabilization of O-acetylserine sulfhydrylase

Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent e nzymes are currently classified into three functional groups and five disti nct structural fold types. The variation within this enzyme group creates a n ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimen sional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly in creasing, but only for relatively few have the folding mechanisms been char acterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonel la typhimurium belongs to the beta -family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holopr otein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, P-31 nuclear magnetic resonan ce, time-resolved fluorescence anisotropy, and photon correlation spectrosc opy indicate that the O-acetylserine sulfhydrylase undergoes extensive disr uption of native secondary and tertiary structure before monomerization. Al so, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are disc ussed in light of the fact that the role of the coenzyme in structural stab ilization varies among the pyridoxal 5'-phosphate-dependent enzymes and doe s not seem to be linked to the particular enzyme fold type.