The Subunit Structure of Tryptophanase I. THE EFFECT OF PYRIDOXAL PHOSPHATE ON THE SUBUNIT STRUCTURE AND PHYSICAL PROPERTIES OF TRYPTOPHANASE

Abstract Homogeneous preparations of apotryptophanase from Escherichia coli required pyridoxal phosphate and K+ or NH4+ ions for activity. The hydrodynamic properties of the apoenzyme vary greatly with the ionic environment and the temperature, and differ markedly from those of holotryptophanase derived from the apoenzyme by interaction with pyridoxal-P. At room temperature and pH 8.0 apotryptophanase exists as a tetramer (mol wt 220,000; s020,w 9.5 S), which dissociates reversibly at low temperature and protein concentration to a dimer (mol wt 110,000; s020,w 6.0 S) when K+ or Na+ is present, but not when these cations are replaced by imidazole. Reversible dissociation to the dimer also occurs at room temperature in the presence of very low concentrations of sodium dodecyl sulfate or when the pH is increased moderately; at higher pH values or sodium dodecyl sulfate concentrations, irreversible dissociation to a monomer (s020,w 3.0 to 3.3 S; mol wt 55,000) occurs. On addition of pyridoxal-P to tetrameric apotryptophanase at 20° in the presence of K+ a major conformational change occurs, as indicated by an increase in s020,w from 9.5 to 10.5 S and a decrease in intrinsic viscosity from 5.55 to 3.80 ml per g without any change in the molecular weight. In contrast to the apoenzyme, the resulting tetrameric holoenzyme does not dissociate to dimer at low temperature and high dilutions; irreversible dissociation to an enzymatically inactive monomer is produced by sodium dodecyl sulfate, but only at concentrations much higher than those required to dissociate the apoenzyme. The equilibrium constant for dissociation of tetrameric to dimeric apotryptophanase increases with decreases in temperature, but the free energy change remains positive at 4°. The enthalpy and entropy changes were calculated to be -50 kcal and -198 e.u., respectively, per mole of tetramer, suggesting the predominantly hydrophobic nature of the bonds between the two dimeric subunits in the tetrameric apoenzyme. On addition of pyridoxal-P to dimeric apoenzyme at 5°, tetrameric holoenzyme is formed. The combination of apoenzyme with pyridoxal-P, measured spectrophotometrically, is a linear function of pyridoxal-P concentration, whereas association of dimer to tetramer shows a sigmoidal relation to the concentration of pyridoxal-P, suggesting a coordinative association of dimeric subunits after their partial saturation with coenzyme. By either criterion, the reaction is complete when 4 moles of coenzyme per mole of tetramer are added. At higher molar ratios pyridoxine-P, pyridoxamine-P, and 5'-phosphopyridoxyltryptophan also promote conversion of dimer to tetramer, indicating that azomethine formation between pyridoxal-P and apotryptophanase is not required to produce the pronounced conformational change that occurs upon holoenzyme formation. The magnitude of this change depends also upon the ionic environment. In the presence of K+, Na+, or imidazole under otherwise identical conditions, tetrameric holotryptophanase shows values for s020,w of 10.5, 10.1, and 9.5 S, respectively, the corresponding value for tetrameric apotryptophanase is 9.5 in each case. The conformational change which results from substitution of Na+ for K+ in the ionic environment is also evidenced at room temperature by a marked change in the spectrum of the holoenzyme, by a marked decrease in the rate of combination of coenzyme with apoenzyme, and, at low temperatures, by a marked reduction in the affinity of the apoenzyme for pyridoxal-P. The four-subunit model of holotryptophanase arrived at from hydrodynamic considerations is confirmed by electron micrographs, which show four subunits apparently arranged in a planar square configuration. The molecular weight calculated by use of the measured diameter of the spherical subunits and the density of the protein agrees well with the value obtained by sedimentation equilibrium measurements.