Role of threonine 101 on the stability of the heme active site of cytochrome P450cam: multiwavelength circular dichroism studies.
The role of the threonine 101 residue that resides close to the heme propionic acid side chain of cytochrome P450cam on the conformational properties of the active site of the enzyme has been investigated by circular dichroism (CD) spectroscopy. Site-specific mutation of the threonine by valine has been carried out that does not affect the size of the residue but significantly alters the hydropathy index. The T101V mutant of cytochrome P450cam showed distinct differences in the CD spectra near the heme region, indicating a subtle effect of the mutation on the properties of the heme active site. Thermal stabilities of the mutant and wild-type enzyme have been studied by temperature dependence of the ellipticity (intensity of the CD band) in the far-UV region for the secondary structure and at different wavelengths in the visible region that arise from the heme moiety for the tertiary structure around the prosthetic group. The thermal unfolding data from variations of the CD intensity at different wavelengths were analyzed using a generalized multistep unfolding model, and two distinct equilibrium intermediate conformational states of the enzyme were identified. The mutation of the T101 residue by valine was found to decrease the thermal stability of both the intermediates in the presence of the substrate. On the other hand, this mutation had no apparent effect on the thermal stability of the enzyme in the absence of the substrate. These results suggested that the threonine residue stabilizes the protein cavity around the heme center in the case of the substrate-bound species, possibly by hydrogen bonding with one of the propionate side chains of the heme moiety. Such hydrogen bonding of the heme propionate with threonine is absent in the substrate-free form of the enzyme.
Manna
Soumen Kanti
SK
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
Mazumdar
Shyamalava
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D002942:Circular Dichroism; D004795:Enzyme Stability; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015183:Restriction Mapping; D013053:Spectrophotometry; D013816:Thermodynamics; D013912:Threonine
D013912:Threonine; D019475:Camphor 5-Monooxygenase