Crystal structures of recombinant human purple Acid phosphatase with and without an inhibitory conformation of the repression loop.
The crystal structure of human purple acid phosphatase recombinantly expressed in Escherichia coli (rHPAP(Ec)) and Pichia pastoris (rHPAP(Pp)) has been determined in two different crystal forms, both at 2.2A resolution. In both cases, the enzyme crystallized in its oxidized (inactive) state, in which both Fe atoms in the dinuclear active site are Fe(III). The main difference between the two structures is the conformation of the enzyme "repression loop". Proteolytic cleavage of this loop in vivo or in vitro results in significant activation of the mammalian PAPs. In the crystals obtained from rHPAP(Ec), the carboxylate side-chain of Asp145 of this loop acts as a bidentate ligand that bridges the two metal atoms, in a manner analogous to a possible binding mode for a phosphate ester substrate in the enzyme-substrate complex. The carboxylate side-chain of Asp145 and the neighboring Phe146 side-chain thus block the active site, thereby inactivating the enzyme. In the crystal structure of rHPAP(Pp), the enzyme "repression loop" has an open conformation similar to that observed in other mammalian PAP structures. The present structures demonstrate that the repression loop exhibits significant conformational flexibility, and the observed alternate binding mode suggests a possible inhibitory role for this loop.
Sträter
Norbert
N
Biotechnologisch-Biomedizinisches Zentrum, Fakultät für Chemie und Mineralogie der Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Jasper
Beate
B
Scholte
Marcel
M
Krebs
Bernt
B
Duff
Anthony P
AP
Langley
David B
DB
Han
Runlin
R
Averill
Bruce A
BA
Freeman
Hans C
HC
Guss
J Mitchell
JM
D000135:Acid Phosphatase / Q000737:chemistry*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins
D006023:Glycoproteins; D011994:Recombinant Proteins; C091137:purple acid phosphatase; D000135:Acid Phosphatase