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Function assignment to conserved residues in mammalian alkaline phosphatases
Umeå University, Faculty of Medicine, Medical Biosciences.
2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 277, no 25, 22992-22999 p.Article in journal (Refereed) Published
Abstract [en]

We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased kcat and increased Km when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxylterminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in kcat, a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors L-Phe and L-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.

Place, publisher, year, edition, pages
2002. Vol. 277, no 25, 22992-22999 p.
URN: urn:nbn:se:umu:diva-20852DOI: 10.1074/jbc.M202298200OAI: diva2:209762
Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2009-03-27
In thesis
1. Structural / functional studies on human alkaline phosphatases
Open this publication in new window or tab >>Structural / functional studies on human alkaline phosphatases
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase (PLAP) has enabled me to perform structural studies aimed at characterizing the properties of human PLAP and tissue-nonspecific AP (TNAP) as paradigms for mammalian APs in general, using site-directed mutagenesis, protein expression, kinetic analysis and computer modeling.

In Paper I, we found that a single critical E429G substitution explains the difference in stability and kinetics between the common allelic variants of PLAP and the D allozyme. In Paper II, we demonstrated the role of residue E429 in PLAP in stabilizing the active site metals, elucidated the distinct roles of residues H153 and H317 in catalysis, and the relative importance of five Cys residues in PLAP. We also discovered the significance of Y367, a unique feature of mammalian APs, for enzyme stability and specific inhibition by amino acids. Paper III focused on the identification and mutagenesis analysis of a novel, non-catalytic peripheral binding site of PLAP that appears to mediate a mitogenic effect of PLAP. This site provides indications that PLAP may function as a fetal growth factor.

The last two papers focus on the TNAP isozyme as paradigm. A deficiency in TNAP activity is the cause of the human disease hypophosphatasia, characterized by rickets, osteomalacia and occasionally epileptic seizures. Paper IV has been able to partially explain the variable expressivity of hypophosphatasia traits by examining site-directed mutants of TNAP and performing kinetic analysis using natural substrates PPi and PLP. Finally, Paper V has clarified the mechanism of inhibition of TNAP by uncompetitive inhibitors L-homoarginine, levamisole and theophylline. We identified residues that confer to TNAP its distinct inhibitory properties. These data have significance for future drug design of specific TNAP inhibitors to therapeutically target TNAP as a way of elevating PPi extracellular level and alleviating pathological bone hypermineralization conditions.

128 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 863
Biomedicine, mutagenesis, enzyme kinetics, computer modeling, crystallography, natural substrates, uncompetitive inhibition, isozymes, gene families, Biomedicin
National Category
Microbiology in the medical area
Research subject
Medical Genetics
urn:nbn:se:umu:diva-136 (URN)91-7305-542-5 (ISBN)
Public defence
2003-11-14, E04, 6E, Norrlands Universitetssjukhus, Umeå, 13:00 (English)
Available from: 2003-10-29 Created: 2003-10-29 Last updated: 2009-03-27Bibliographically approved

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