Biosynthetic l-threonine deaminase from Bacillus stearothermophilus: I. Catalytic and regulatory properties

Abstract

A biosynthetic l-threonine deaminase has been purified approximately sixtyfold from the obligate thermophile Bacillus stearothermophilus. The enzyme is partially stabilized against heat inactivation at 70 ° in the presence of either threonine or pyridoxal phosphate. The deaminase has a temperature optimum near 65 ° and a pH optimum of 9.0–9.5 in glycine-NaOH buffer. The sensitivity of the deaminase to feedback inhibition by l-isoleucine decreases as the assay temperature is increased from 30 to 70 °. The inhibition is also antagonized at high pH without loss of catalytic activity. These results suggest that the enzyme possesses separate binding sites for both substrate and end-product. Enzyme activity is stimulated in the presence of K +, Na +, and Li +. Of these monovalent cations only Na + increased both enzyme activity and sensitivity to isoleucine inhibition. At 55 °, substrate saturation curves indicate hyperbolic kinetics for threonine binding. However, in the presence of either isoleucine or Na + cooperative homotropic effects for theronine were observed. Inhibition curves with increasing isoleucine concentrations at 55 ° also indicated sigmoidal inhibition kinetics. The kinetic data suggest that the enzyme molecule contains multiple binding sites for both threonine and isoleucine. A molecular weight of approximately 200,000 was estimated for the deaminase.