Looking for residues involved in the muscle acylphosphatase catalytic mechanism and structural stabilization: role of Asn41, Thr42, and Thr46.
Abstract: Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were purified by affinity chromatography and investigated as compared to the wild-type enzyme. The specific activity and substrate affinity of Thr42 and Thr46 mutants were not significantly affected. On the contrary, Asn41 mutants showed a residual negligible activity (about 0.05-0.15% as compared to wild-type enzyme), though maintaining an unchanged binding capability of both substrate and inorganic phosphate, an enzyme competitive inhibitor. According to the 1H nuclear magnetic resonance spectroscopy and circular dichroism results, all mutants elicited well-constrained native-like secondary and tertiary structures. Thermodynamic parameters, as calculated from circular dichroism data, demonstrated a significantly decreased stability of the Thr42 mutant under increasing temperatures and urea concentrations. The reported results strongly support a direct participation of Asn41 to the enzyme catalytic mechanism, indicating that Asn41 mutants may well represent a useful tool for the investigation of the enzyme physiological function by the negative dominant approach.
Publication Date: 1996-06-04 PubMed ID: 8679533DOI: 10.1021/bi952900bGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research aimed to study the role of specific residues – Asn41, Thr42, and Thr46 – in the structural stabilization and catalytic mechanism of muscle acylphosphatase. It found that Asn41 is crucial for the enzyme’s catalytic action, while changing the Thr42 significantly decreased the enzyme’s stability.
Methodology
- The study focused on Asn41, Thr42, and Thr46, residues found in muscle and erythrocyte acylphosphatases. It noted these residues have a close spatial relationship to Arg23, the substantial substrate binding site.
- Researchers investigated these residues’ catalytic and structural roles by preparing several gene mutants. The mutated genes included Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln; all of these were prepared through oligonucleotide-directed mutagenesis.
- These mutated genes were cloned and expressed in Escherichia coli. The derived mutant enzymes were then purified using affinity chromatography for comparison with the wild-type enzyme.
Findings
- The mutants Thr42 and Thr46 did not significantly affect the enzyme’s activity or substrate affinity.
- The Asn41 mutants, however, exhibited a significantly reduced activity (around 0.05-0.15% compared to the wild-type enzyme), despite maintaining the same binding capability for both the substrate and inorganic phosphate.
- All mutants formed well-constrained secondary and tertiary structures, similar to the native enzyme, according to results gathered using 1H nuclear magnetic resonance spectroscopy and circular dichroism.
- However, the Thr42 mutant exhibited significantly decreased stability under increasing temperatures and urea concentrations, as determined based on circular dichroism data.
Conclusion
- The study concludes that Asn41 plays an essential role in the catalytic mechanism of the enzyme, making its mutants a useful tool for studying the enzyme’s physiological function using the negative dominant approach.
- The research highlights the importance of understanding these residues in assessing the performance and stability of the acylphosphatase enzyme, which can have implications in various biological and medical applications.
Cite This Article
APA
Taddei N, Stefani M, Magherini F, Chiti F, Modesti A, Raugei G, Ramponi G.
(1996).
Looking for residues involved in the muscle acylphosphatase catalytic mechanism and structural stabilization: role of Asn41, Thr42, and Thr46.
Biochemistry, 35(22), 7077-7083.
https://doi.org/10.1021/bi952900b Publication
Researcher Affiliations
- Department of Biochemical Sciences, University of Florence, Italy.
MeSH Terms
- Acid Anhydride Hydrolases / chemistry
- Acid Anhydride Hydrolases / genetics
- Acid Anhydride Hydrolases / metabolism
- Animals
- Asparagine / chemistry
- Base Sequence
- Binding Sites
- Catalysis
- Circular Dichroism
- Enzyme Stability
- Horses
- Kinetics
- Magnetic Resonance Spectroscopy
- Models, Molecular
- Molecular Sequence Data
- Muscles / enzymology
- Mutagenesis, Site-Directed
- Protein Denaturation
- Protein Structure, Secondary
- Protein Structure, Tertiary
- Recombinant Fusion Proteins / isolation & purification
- Recombinant Fusion Proteins / metabolism
- Substrate Specificity
- Thermodynamics
- Threonine / chemistry
Citations
This article has been cited 11 times.- Fusco G, Bemporad F, Chiti F, Dobson CM, De Simone A. The role of structural dynamics in the thermal adaptation of hyperthermophilic enzymes.. Front Mol Biosci 2022;9:981312.
- Dagan S, Hagai T, Gavrilov Y, Kapon R, Levy Y, Reich Z. Stabilization of a protein conferred by an increase in folded state entropy.. Proc Natl Acad Sci U S A 2013 Jun 25;110(26):10628-33.
- Pellistri F, Bucciantini M, Relini A, Nosi D, Gliozzi A, Robello M, Stefani M. Nonspecific interaction of prefibrillar amyloid aggregates with glutamatergic receptors results in Ca2+ increase in primary neuronal cells.. J Biol Chem 2008 Oct 31;283(44):29950-60.
- Calamai M, Chiti F, Dobson CM. Amyloid fibril formation can proceed from different conformations of a partially unfolded protein.. Biophys J 2005 Dec;89(6):4201-10.
- Maxwell KL, Wildes D, Zarrine-Afsar A, De Los Rios MA, Brown AG, Friel CT, Hedberg L, Horng JC, Bona D, Miller EJ, Vallée-Bélisle A, Main ER, Bemporad F, Qiu L, Teilum K, Vu ND, Edwards AM, Ruczinski I, Poulsen FM, Kragelund BB, Michnick SW, Chiti F, Bai Y, Hagen SJ, Serrano L, Oliveberg M, Raleigh DP, Wittung-Stafshede P, Radford SE, Jackson SE, Sosnick TR, Marqusee S, Davidson AR, Plaxco KW. Protein folding: defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins.. Protein Sci 2005 Mar;14(3):602-16.
- Chiti F, Calamai M, Taddei N, Stefani M, Ramponi G, Dobson CM. Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases.. Proc Natl Acad Sci U S A 2002 Dec 10;99 Suppl 4(Suppl 4):16419-26.
- Chiti F, Taddei N, Stefani M, Dobson CM, Ramponi G. Reduction of the amyloidogenicity of a protein by specific binding of ligands to the native conformation.. Protein Sci 2001 Apr;10(4):879-86.
- Cecchi C, Liguri G, Pieri A, Degl'Innocenti D, Nediani C, Fiorillo C, Nassi P, Ramponi G. Interaction between acylphosphatase and SERCA in SH-SY5Y cells.. Mol Cell Biochem 2000 Aug;211(1-2):95-102.
- Paoli P, Camici G, Manao G, Giannoni E, Ramponi G. Acylphosphatase possesses nucleoside triphosphatase and nucleoside diphosphatase activities.. Biochem J 2000 Jul 1;349(Pt 1):43-9.
- Chiti F, Taddei N, Bucciantini M, White P, Ramponi G, Dobson CM. Mutational analysis of the propensity for amyloid formation by a globular protein.. EMBO J 2000 Apr 3;19(7):1441-9.
- Paoli P, Cirri P, Camici L, Manao G, Cappugi G, Moneti G, Pieraccini G, Camici G, Ramponi G. Common-type acylphosphatase: steady-state kinetics and leaving-group dependence.. Biochem J 1997 Oct 1;327 ( Pt 1)(Pt 1):177-84.
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