Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis.
Aminoacylase-1 from pig kidney (pAcy1) catalyzes the highly stereoselective acylation of amino acids, a useful conversion for the preparation of optically pure N-acyl-l-amino acids. The kinetic of this thermodynamically controlled conversion is determined by maximal velocities for synthesis (V(mS)) and hydrolysis (V(mH)) of the N-acyl-l-amino acid. To investigate which parameter affects maximal velocities, we focused on the proton acceptor potential of the catalytic base, E146, and studied the influence of the active site architecture on its contribution to the pKa of residue E146. The modeled structure of pAcy1 identified residue D346 as having the strongest impact on the electrostatic features of the catalytic base. Substitutions of D346 generally decreased enzymatic activities but also altered both the pH-dependency of hydrolytic activity and the V(mS)/V(mH) ratio of pAcy1. A reduced theoretical pKa value and a lowered experimental pH optimum of hydrolytic rates for the D346A mutant were associated with a 9-fold increase in V(mS)/V(mH). This supports the importance of electrostatic contributions of D346 to the acid-base properties of E146 and demonstrates for the first time the possibility of engineering the V(mS)/V(mH) ratio of pAcy1.
Wardenga
Rainer
R
Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, Greifswald University, Germany.
Lindner
Holger A
HA
Hollmann
Frank
F
Thum
Oliver
O
Bornscheuer
Uwe
U
D000215:Acylation; D000581:Amidohydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000596:Amino Acids / Q000096:biosynthesis* / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013552:Swine
D000596:Amino Acids; D000581:Amidohydrolases; C019672:aminoacylase I; D009584:Nitrogen