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Structural and functional analysis of a non-catalytic binding site in human placental alkaline phosphatase
Umeå University, Faculty of Medicine, Department of Medical Biosciences.
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(English)Manuscript (Other academic)
URN: urn:nbn:se:umu:diva-20853OAI: diva2:209779
Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2012-02-01
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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Kozlenkov, AlexeyStigbrand, Torgny
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