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Protein Science (2007), 16:441-448. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Important roles of hydroxylic amino acid residues in the function of Bacillus subtilis adenylosuccinate lyase

Mark L. Segall, Meghan A. Cashman, and Roberta F. Colman

Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA

(RECEIVED November 2, 2006; FINAL REVISION December 6, 2006; ACCEPTED December 7, 2006)

Thr93, Ser94, Thr140, and Ser306 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 Vmax of wild-type ASL with a 10-fold increase in Km for adenylosuccinate. S94A has 65% of the Vmax of wild-type ASL with little change in Km. T140A exhibits 0.03% of the activity of wild-type enzyme with an 11-fold increase in Km. S306A has 0.4% of the Vmax of wild-type ASL with a sevenfold increase in Km. Measurements of the pH-Vmax profile reveal a pK2 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. Thr93 may orient adenylosuccinate optimally for catalysis, while Ser94 stabilizes protonated His89, a determinant of pK2. Thr140 may, through hydrogen bonding, interact with Asn270, an amino acid essential for catalysis. Ser306 may be involved in a hydrogen bond network that ultimately stabilizes protonated His68, 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.

Keywords: adenylosuccinate lyase; purine biosynthesis; site-directed mutagenesis; hydrogen bonding


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S. Sivendran, M. L. Segall, P. C. Rancy, and R. F. Colman
Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase
Protein Sci., August 1, 2007; 16(8): 1700 - 1707.
[Abstract] [Full Text] [PDF]


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