Mammalian alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino acid microenvironment.

In mammalian alkaline phosphatase (AP) dimers, the N-terminus of one monomer embraces the other, stretching toward its active site. We have analyzed the role of the N-terminus and its microenvironment in determining the enzyme stability and catalysis using human placental (PLAP) and tissue-nonspecific AP (TNAP) as paradigms. Deletion of nine amino acid (aa) residues in PLAP reduced its AP activity and heat stability, while deletion of 25 aa resulted in an inactive enzyme. In turn, deletion of five and nine N-terminal aa in TNAP reduced and abolished AP activity, respectively. The N-terminal aa deletions in both isozymes affected the rate of substrate catalysis (k(cat)), with an only minor effect on the Michaelis constant (K(m)) explained by decelerated intramolecular transition rates in the active site. Arg370 in PLAP, and the corresponding Arg374 in TNAP, critically control the structure and function of the enzymes, but the Glu6-Arg370 bond predicted by the PLAP crystal structure appeared to be irrelevant with respect to PLAP stability or catalysis. Structural disruption was also noted in [R374A]TNAP, [Delta5]TNAP, [Delta9]TNAP, and [Delta25]TNAP using a panel of 19 anti-TNAP antibodies illustrating the structural role of the N-terminus. Our data reveal that the N-terminal alpha-helical folding is more crucial for the structural stability of the second monomer in TNAP than in PLAP. The correct folding of the N-terminus and of interacting loops in its immediate environment is essential for overall structural integrity and for execution of intramolecular transitions during enzyme catalysis. These findings provide a mechanistic interpretation for loss-of-function mutations of N-terminal TNAP residues in cases of hypophosphatasia.