The large size and the multimeric nature of Asp-NAT called for an investigation on the stability characteristics of this complex. CHAPS and NaCl were used in increasing concentrations in the treatment medium to perturb the Asp-NAT complex. Enriched Asp-NAT preparations from a DEAE column were used in these studies in view of its higher enzyme activity. The results in Figure 6 show that CHAPS has a biphasic effect on Asp-NAT stability (Fig. 6A) as revealed by pre-treatment (0-4 oC, 1h). Asp-NAT activity decreased to 40% of the control at 12 mM CHAPS and 10% of the control at 20 mM CHAPS. Removal of excess CHAPS by dialysis had no restoring effect on the enzyme activity after pre-treatment at >12 mM CHAPS. Although NaCl decreased Asp-NAT
activity at concentrations greater than 0.15 M, removal of excess NaCl by dialysis reversed the effect up to 1 M NaCl (Fig. 6B).
CHAPS, mM NaCl, M
Figure 6. Asp-NAT activity in the presence of varying concentrations of CHAPS (A) and NaCl (B). Relative activity (%) with respect to 1 mM CHAPS in Fig. A, 0.15 M NaCl in Fig. B is represented in the y-axis. Enzyme activity was more or less fully restored by dialysis (squares) after treating in high concentrations of NaCl (Fig. B).
Pre-treatment for 1h in 0.15 M, 0.5 M and 2.0 M NaCl media and HPLC with a size exclusion column (~850 kD cut-off) gave unimodal distribution of Asp-NAT activity in the eluted fractions (Fig. 7A). Enzyme activity was somewhat decreased at 0.5 and 2.0 M NaCl, probably due to incomplete dialysis. However, pre-treatment with 10 mM CHAPS (as opposed to 1 mM CHAPS in the buffer) and HPLC size exclusion chromatography resulted in significant loss of activity, which was irreversible even after dialysis. The distribution pattern was also different with a right shift (Fig. 7B) and increased flatness, as compared to the control (1 mM CHAPS pre-treatment). These results are consistent with the possibility that some subunits that contribute or regulate the enzyme activity are separated from the complex during the chromatography, which results in the loss of enzyme activity. That these enzyme components are held primarily by hydrophobic interactions is indicated by the sensitivity to CHAPS, but not to increasing ionic strength.
The steady state kinetic parameters of Asp-NAT are presented in Table 2. Between the two substrates, Asp-NAT has an order of magnitude higher affinity (Km) toward acetyl CoA than L-Asp. Both the products, NAA and CoA have inhibitory effect on Asp-NAT activity. This enzyme was specific toward L-Asp and showed <3% activity against L-Glu, L-Asn and L-Gln.
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