Abstract
Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.
Highlights
Pyridoxal 5Ј-phosphate (PLP)1-dependent enzymes have been classified into the ␣, , and ␥ families based on the chemical characteristics of their enzymatic activities [1]
Threedimensional structures have been reported for about 30 PLPdependent enzymes [3], and the folding mechanism has been investigated in some detail for less than a dozen
Despite the well known uncertainty in the quantitative evaluation of secondary structure content from CD spectra, the calculated values for holo-O-acetylserine sulfhydrylase (OASS) are in good agreement with the crystallographic data (Table II)
Summary
Pyridoxal 5Ј-phosphate (PLP)1-dependent enzymes have been classified into the ␣, , and ␥ families based on the chemical characteristics of their enzymatic activities [1]. This enzyme group has been organized on the basis of sequence. Tryptophan synthase, threonine deaminase, and O-acetylserine sulfhydrylase (OASS) belong to the -family and fold type II. These enzymes catalyze -replacement and/or -elimination reactions, sharing similar catalytic pathways. A detailed investigation into the folding mechanism of the PLP-dependent enzymes belonging to the same or different families can provide a rare opportunity to uncover the specific structure and function determinants encoded in the primary sequence. The conformational properties of OASS have been investigated in the presence and absence of bound PLP and during the catalytic reaction by measuring the steady-
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