Important roles of hydroxylic amino acid residues in the function of Bacillus subtilis adenylosuccinate lyase

Thr 93 , Ser 94 , Thr 140 , and Ser 306 are conserved in all adenylosuccinate lyases (ASL) and are close to other amino acids previously identified by mutagenesis as being in the active site. To test their involvement in the enzyme's function, each of these amino acids was replaced by alanine. All the mutants exhibit circular dichroism spectra which are similar to that of wild‐type enzyme, indicating there is no appreciable change in secondary structure. T93A exhibits 0.5% of the V max of wild‐type ASL with a 10‐fold increase in K m for adenylosuccinate. S94A has 65% of the V max of wild‐type ASL with little change in K m . T140A exhibits 0.03% of the activity of wild‐type enzyme with an 11‐fold increase in K m . S306A has 0.4% of the V max of wild‐type ASL with a sevenfold increase in K m . Measurements of the pH‐V max profile reveal a pK 2 value for S94A of 7.83 and S306A of 7.65, in contrast to 8.24 for the wild‐type enzyme and 8.42 for T93A. Thr 93 may orient adenylosuccinate optimally for catalysis, while Ser 94 stabilizes protonated His 89 , a determinant of pK 2 . Thr 140 may, through hydrogen bonding, interact with Asn 270 , an amino acid essential for catalysis. Ser 306 may be involved in a hydrogen bond network that ultimately stabilizes protonated His 68 , which is probably the general acid in the reaction of enzyme with substrate. The results of this paper demonstrate the importance in the catalytic function of ASL of hydrogen bonds and hydrogen bonding networks involving serine and threonine.