Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants.
Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.
Truong
Ngoc Thanh
NT
Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany.
Naseri
Joseph Itor
JI
Vogel
Andreas
A
Rompel
Annette
A
Krebs
B
B
D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002412:Cations; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D027805:Phaseolus / Q000201:enzymology*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D015032:Zinc / Q000737:chemistry*
D002412:Cations; D006023:Glycoproteins; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D015032:Zinc