Modifications in a flexible surface loop modulate the isozyme-specific properties of mammalian alkaline phosphatases.
We have analyzed to what extent the surface loop domain of alkaline phosphatases (APs) is responsible for isozyme-specific functional properties. Unique AatII and RsrII restriction sites were introduced by site-directed mutagenesis at identical positions in murine tissue-nonspecific AP (TNAP) and human placental AP (PLAP) cDNAs to allow the homologous exchange of the loop domain of the TNAP (T domain) and PLAP (P domain) isozymes and the generation of the reciprocally chimeric molecules PLAP-T and TNAP-P. The introduction of the T loop into PLAP reduced the heat stability of PLAP-T to almost that of TNAP. The domain substitution was accompanied by a conformational change that resulted in the loss of immune reactivity with four of 17 epitope-mapped anti-PLAP monoclonal antibodies. The T and P loops provided stabilization to the side chain of specific uncompetitive AP inhibitors. The introduction of the T domain also conferred collagen-binding properties to PLAP-T accounting for half of the binding affinity of TNAP for collagen, while not affecting PLAP binding to IgG. Our data indicate that the surface loop determines overall enzyme stability, differs conformationally in the various isozymes, and modulates catalytic parameters in the presence of protein ligands, thus, accounting in part for isozyme-specific protein interactions.
Bossi
M
M
La Jolla Cancer Research Foundation, California 92037.
Hoylaerts
M F
MF
Millán
J L
JL
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase