Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

[1]  A. Guerrieri,et al.  A novel approach for the selective analysis of l-lysine in untreated human serum by a co-crosslinked l-lysine-α-oxidase/overoxidized polypyrrole bilayer based amperometric biosensor. , 2018, Bioelectrochemistry.

[2]  A. Arinbasarova,et al.  Kinetic characteristics of L-lysine α- oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D: Substrate specificity and allosteric effects , 2016, Biochemistry and biophysics reports.

[3]  Antonio Guerrieri,et al.  Quantification of l-lysine in cheese by a novel amperometric biosensor. , 2015, Food chemistry.

[4]  T. Cataldi,et al.  A novel amperometric biosensor based on a co-crosslinked L-lysine-α-oxidase/overoxidized polypyrrole bilayer for the highly selective determination of L-lysine. , 2013, Analytica chimica acta.

[5]  V. Pokrovsky,et al.  Enzymatic properties and anticancer activity of L-lysine &agr;-oxidase from Trichoderma cf. aureoviride Rifai BKMF-4268D , 2013, Anti-cancer drugs.

[6]  Edinburgh Research Explorer Cooperative Binding , 2022 .

[7]  K. V. Makrushin,et al.  Isolation and properties of L-lysine-α-oxidase from the fungus Trichoderma cf. aureoviride RIFAI VKM F-4268D , 2012, Microbiology.

[8]  Kevin W Plaxco,et al.  Re-engineering electrochemical biosensors to narrow or extend their useful dynamic range. , 2012, Angewandte Chemie.

[9]  Giuseppe Palleschi,et al.  Employing the metabolic "branch point effect" to generate an all-or-none, digital-like response in enzymatic outputs and enzyme-based sensors. , 2012, Analytical chemistry.

[10]  A. A. Efremova,et al.  L-Amino acid oxidases: Properties and molecular mechanisms of action , 2011, Biomeditsinskaia khimiia.

[11]  T. Cataldi,et al.  The kinetic and analytical behaviours of an l-lysine amperometric biosensor based on lysine oxidase immobilised onto a platinum electrode by co-crosslinking , 2007 .

[12]  D. Schomburg,et al.  The structure of a bacterial L-amino acid oxidase from Rhodococcus opacus gives new evidence for the hydride mechanism for dehydrogenation. , 2007, Journal of molecular biology.

[13]  T. Berezov,et al.  L-Lysine α-Oxidase: Physicochemical and Biological Properties , 2002, Biochemistry (Moscow).

[14]  T. Berezov,et al.  Isoforms of L-lysine α-oxidase fromTrichoderma sp. , 1996, Bulletin of Experimental Biology and Medicine.

[15]  S. Reiken,et al.  The effect of lysine deprivation on leukemic blood , 1992, Amino Acids.

[16]  K. Soda,et al.  A new antitumor enzyme, L-lysine alpha-oxidase from Trichoderma viride. Purification and enzymological properties. , 1980, The Journal of biological chemistry.

[17]  David Gough,et al.  Membrane-covered, rotated disk electrode , 1979 .

[18]  K. Soda,et al.  Occurrence of a Novel Enzyme, L-Lysine Oxidase with Antitumor Activity in Culture Extract of Trichoderma viride , 1979 .

[19]  D. S. Page,et al.  L-amino acid oxidase. II. Deuterium isotope effects and the action mechanism for the reduction of L-amino acid oxidase by L-leucine. , 1971, Biochimica et biophysica acta.

[20]  R. V. Van Etten,et al.  L-amino-acid oxidase. I. Effect of pH. , 1969, Biochimica et biophysica acta.

[21]  J P Changeux,et al.  On the nature of allosteric transitions: implications of non-exclusive ligand binding. , 1966, Journal of molecular biology.

[22]  J. Changeux,et al.  ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. , 1965, Journal of molecular biology.

[23]  P. Fitzpatrick.  Oxidation of amines by flavoproteins. , 2010, Archives of biochemistry and biophysics.

[24]  Laurence A. Nafie,et al.  A combined theoretical and experimental study of the structure and vibrational absorption, vibrational circular dichroism, Raman and Raman optical activity spectra of the L-histidine zwitterion , 2008 .

[25]  Jonathan M. Cooper,et al.  Biosensors : a practical approach , 2004 .

[26]  P. Zambonin,et al.  Amperometric sensor for choline and acetylcholine based on a platinum electrode modified by a co-crosslinked bienzymic system , 1995 .

[27]  P. Patel.  Enzymes as diagnostic tools , 1995 .

[28]  E. Weber,et al.  L‐Lysine α‐oxidase from Trichoderma viride i4. Purification and characterization , 1994 .

[29]  D. Schomburg,et al.  L-Amino-acid oxidase , 1993 .

[30]  A. F. Albantov.  Reducing the influence of sample oxygen in biosensor design , 1992 .

[31]  F. Wedler.  Enzyme kinetics: Behavior and analysis of rapid equilibrium and steady‐state enzyme systems. Author: Irwin H. Segal (University of California, Davis). Published by Wiley‐Interscience, New York, 1975. Price: $24.50. No. of pages: 957 , 1976 .