| Abstrakt: |
Acetylation of Escherichia coli aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) with acetic anhydride or N-hydroxysuccinimide acetate resulted in the alteration of catalytic and regulatory properties as follows. At pH 7.0, 2-fold activation was observed in 30 min, whereas at pH 8.5 the activity of the acetylated enzyme was lower than that of the native enzyme throughout the range of substrate concentrations tested, while maintaining the Vmax unchanged. The Hill coefficient values of the substrate saturation curves were also altered under both pH conditions to an appreciable extent toward higher values. The enzyme activity's requirement for divalent metal ions increased at both pH 7.0 and 8.5. In particular the ratio of the activities in the presence vs. absence of Mg2+ reached as high as 84.5 at the latter pH. Inspection of the acetylation-induced conformational change by difference absorption spectroscopy revealed that a red shift occurred in the ultraviolet region. Chemical analyses, including high-performance liquid chromatography, of the acetylated residues revealed that approximately three amino groups per subunit were acetylated concomitant with a 2.1-fold activation and that the acetylation site was restricted to a relatively specific region of the enzyme molecule. Acylation of the enzyme with other acid anhydrides such as n-butyric and propionic anhydrides also increased the activity at pH 7.0, although to a lesser extent. |
