An Australian patient with
autism was found to be heterozygous for two mutations in the gene encoding
adenylosuccinate lyase (ASL), resulting in the
protein mutations E80D and D87E. The patient's mother carried only the E80D mutation. The equivalent positions are 62 and 69 in Bacillus subtilis ASL. Although both human and B. subtilis
enzymes normally have Asp at position 87 (or 69), the B. subtilis ASL has Ile and Asp at 62 and 65, respectively, whereas human ASL has Glu and Arg at the equivalent positions. We have constructed, expressed, and purified the double mutant I62E/D65R as a "humanized" normal B. subtilis
enzyme to compare with
enzymes with a single mutation at position 62 (I62D/D65R), at position 69 (I62E/D65R/D69E), or at both positions (I62D/D65R/D69E). V(max) for conversion of
adenylosuccinate to
AMP and
fumarate is 0.57 micromol/min/mg for I62E/D65R, 0.064 micromol/min/mg for I62D/D65R, 0.27 micromol/min/mg for I62E/D65R/D69E, and 0.069 micromol/min/mg for I62D/D65R/D69E. The K(m) for
adenylosuccinate is elevated in the X62D mutants, and I62D/D65R is the least stable of these ASLs at 37 degrees C. The CD spectra of mutant and wild type
enzymes are similar; thus, there are no appreciable structural changes. Clearly the Asp(62) causes the most drastic effect on ASL function, whereas the Glu(69) mutation produces only modest change. These results emphasize the importance of expanding tests for
ASL deficiency to individuals with developmental delay of any severity, including individuals with
autistic spectrum disorder. This study further demonstrates the usefulness of the B. subtilis ASL as a model to mimic the defective
enzyme in
ASL deficiency.