Acid-Base Catalyst Discriminates between a Fructose 6-Phosphate Aldolase and a Transaldolase

作者：Stellmacher Lena; Sandalova Tatyana; Leptihn Sebastian; Schneider Gunter; Sprenger Georg A; Samland Anne K^*

来源：ChemCatChem, 2015, 7(19): 3140-3151.

DOI：10.1002/cctc.201500478

• C-C coupling
• enzyme catalysis
• general acid/base
• protein engineering
• structure elucidation

The residues responsible for binding the catalytic water molecule were interchanged between the closely related enzymes fructose 6-phosphate aldolase A (FSAA) and transaldolase B (TalB) from Escherichia coli. In FSAA, this water molecule is bound by hydrogen bonds to the side chains of three residues (Gln59, Thr109 and Tyr131), whereas in TalB only two residues (Glu96 and Thr156) participate. Single and double variants were characterised with respect to fructose 6-phosphate aldolase and transaldolase activity, stability, pH dependence of activity, pK(a) value of the essential lysine residue and their three dimensional structure. The double variant TalB(E96QF178Y) showed improved aldolase activity with an apparent k(cat) of 4.3s(-1). The experimentally determined pK(a) values of the catalytic lysine residue revealed considerable differences: In FSAA, this lysine residue is deprotonated at assay conditions (pK(a)5.5) whereas it is protonated in TalB (pK(a)9.3). Hence, a deprotonation of the catalytic lysine residue, which is a prerequisite for an efficient nucleophilic attack in TalB, is not necessary in FSAA. Based upon these results, we propose a new mechanism for FSAA with Tyr131 as general acid.

• 出版日期2015-10-5