Influence of cofactor pyridoxal 5'-phosphate on reversible high-pressure denaturation of isolated beta 2 dimer of tryptophan synthase bienzyme complex from Escherichia coli.
High hydrostatic pressure has been shown to cause reversible dissociation of the isolated apo beta 2 dimer of tryptophan synthase from Escherichia coli into enzymatically inactive monomers [Seifert, T., Bartholmes, P., & Jaenicke, R. (1982) Biophys. Chem. 15, 1-8]. Addition of the coenzyme pyridoxal 5'-phosphate affects the structural stability, as well as the kinetics of dissociation and deactivation. The apo beta 2 dimer is deactivated faster than the holoenzyme by a factor of 10. The midpoints of the corresponding equilibrium transition curves are observed at 690 and 870 bar, respectively. As shown by hybridization of native and chemically modified beta chains, the loss of enzymatic activity is accompanied by subunit dissociation. An additional deactivating effect is produced by the pressure-induced release of the cofactor from the holoenzyme. Renaturation after decompression has been monitored by circular dichroism and intrinsic fluorescence emission. Alterations of the dichroic absorption at 222 nm reflect the recovery of the native secondary structure, while tryptophan fluorescence represents a specific probe for the native tertiary structure in the immediate neighborhood of the active center of the enzyme. By application of both methods to monitor the reconstitution of the apo beta 2 dimer, two first-order processes may be separated along the time scale. The faster phase (k1 = 1.2 X 10(-2) s-1) yields a "structured monomer" with 85% native secondary structure and the tryptophan side chain buried in its native hydrophobic environment. As indicated by sodium borohydride reduction, this intermediate is able to interact with the coenzyme pyridoxal 5'-phosphate in the correct way; however, it does not show enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D001051:Apoenzymes; D004926:Escherichia coli / Q000201:enzymology*; D006874:Hydrostatic Pressure; D007700:Kinetics; D046911:Macromolecular Substances; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013329:Structure-Activity Relationship; D014367:Tryptophan Synthase / Q000378:metabolism*
D001051:Apoenzymes; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase