Modifications in a flexible surface loop modulate the isozyme-specific properties of mammalian alkaline phosphatases.

M Bossi,M F Hoylaerts,J L Millán

doi:10.1016/s0021-9258(19)74407-x

Abstract

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.

Highlights

From the $LaJolla Cancer Research Foundation, La Jolla, California 92037 and the
Our data indicate that the surface loop determines First, while the bacterial alkaline phosphatases (APs) is released after synthesis to overall enzyme stability, differs conformationally in the periplasmic space, mammalian APs are anchored to the the various isozymes, and modulatecsatalytic param- exterior of the cytoplasmic membrane via a phosphatidylinoeters in the presence of protein ligands, accounts-itol glycan moiety (Low and Saltiel, 1988)
In order to study the function of this surface loop, we focused on two isozymes with considerably different biochemical properties; i.e. on one hand murine TNAP which is very heat labile, binds to collagen type I, 11,and X and is inhibited uncompetitively by L-homoarginine and on the other hand human PLAPw, hich is extremely heat stable, interacts with the Fc region of IgG and is inhibited uncompetitively by t-Phe, L-Leu, and related peptides

Summary

Introduction

From the $LaJolla Cancer Research Foundation, La Jolla, California 92037 and the 1991).The surface loop of PLAP/GCAP that harbors residue 429 plays an important role in determining the accessibility and stability of the inhibitor in the active site duringuncompetitive inhibition(Hoylaerts et al, 1992a).

Results

Conclusion