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Protein Science (2004), 13:913-924. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations

Barbara Pioselli1,3, Stefano Bettati2,3, Tatyana V. Demidkina4, Lyudmila N. Zakomirdina4, Robert S. Phillips5 and Andrea Mozzarelli1,3

1 Department of Biochemistry and Molecular Biology,
2 Department of Public Health, and
3 National Institute for the Physics of Matter, University of Parma, 43100 Parma, Italy
4 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia
5 Department of Chemistry and Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA

(RECEIVED October 27, 2003; FINAL REVISION December 4, 2003; ACCEPTED December 4, 2003)



Abstract

The pyridoxal 5'-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold–eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix.

Keywords: protein immobilization; pyridoxal 5'-phosphate; catalysis; silica gels; conformational selection

Abbreviations: TPL, Tyrosine phenol-lyase • Trpase, tryptophan indole-lyase • PLP, pyridoxal 5'-phosphate • SOPC, S-(o-Nitrophenyl)-L-cysteine • S-Me-Cys, S-methyl-L-cysteine • 3-F-Tyr, 3-fluoro-L-tyrosine • 4-OH-pyr, 4-hydroxy-pyridine • OIA, oxindolyl-L-alanine • TMOS, tetramethyl orthosilicate

Reprint requests to: Andrea Mozzarelli, Department of Biochemistry and Molecular Biology, University of Parma, Via Parco delle Scienze 23/A, 43100 Parma, Italy; e-mail: biochim{at}unipr.it; fax: 39-0521-905151.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03492904.


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