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Residues determining the specific binding of uncompetitive inhibitors to mammalian alkaline phosphatases
Umeå University, Faculty of Medicine, Department of Medical Biosciences.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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(English)Manuscript (Other academic)
Abstract [en]

Recent data have suggested that the activity of human tissue-nonspecific alkaline phosphatase (TNAP) could be targeted therapeutically to ameliorate soft-tissue ossification abnormalities resulting from insufficient production of inorganic pyrophosphate (1). Thus, understanding the mechanism of action and precise binding site for inhibitors of TNAP function is paramount. We compared the modeled three-dimensional structure of TNAP with the 3D structure of human placental alkaline phosphatase (PLAP), and identified the residues that differ between these two isozymes within a 12 Å radius of the active site. We then used site-directed mutagenesis to substitute TNAP residues to their respective homologues in PLAP or in the related germ cell (GCAP) isozyme or to Ala. In addition, we mutagenized most of the corresponding residues in PLAP to their TNAP homologues, and studied the role of a conserved residue Y371 in TNAP using the A371 mutation. All mutants were characterized for their sensitivity towards the uncompetitive inhibitors Lhomoarginine (L-hArg), levamisole, theophylline and L-phenylalanine. We found that the identity of residue108 in TNAP largely determines the specificity of inhibition by LhArg. The conserved Tyr-371 is also necessary for binding of L-hArg. The selectivity of inhibition by L-Phe was determined by the identity of residues 108 and 109 in TNAP (or corresponding residues 107 and 108 in PLAP). In contrast, the binding of levamisole to TNAP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. Substitutions H434E (as in PLAP), H434Q (as in chicken TNAP), H434S (as in the intestinal isozyme), H434G (as in GCAP), or H434A, all lead to a significant decrease in inhibition. The reciprocal E429H mutation in PLAP improved the inhibition by levamisole by 10-fold. The main determinant of sensitivity to theophylline is His-434. The H434E substitution (as in PLAP) causes a 50-fold reduction in inhibition, making TNAP even less inhibited than wt PLAP. The reciprocal E429H mutation in PLAP improves the Ki for theophylline inhibition 30 times, close to the level of wt TNAP. Interestingly, theophylline inhibition in TNAP could be further improved by introducing an E108F mutation. Thus, we have clarified the location of the binding sites for all three TNAP inhibitors and we have also been able to exchange inhibitor specificities between TNAP and PLAP. These data will help us in drug design efforts aimed at discovering novel and better inhibitors of TNAP for clinical use.

URN: urn:nbn:se:umu:diva-20868OAI: diva2:209790
Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2011-03-30
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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