Kinetics of the Interaction of Peptidases with Substrates and Modifiers
This chapter is dedicated to reviewing, commenting and illustrating with examples kinetic tools that are essential to the study of peptidases interacting with their substrates, inhibitors and activators. Kinetic characterization starts with the measuremen
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Kinetics of the Interaction of Peptidases with Substrates and Modifiers Antonio Baici, Marko Novinec, and Brigita Lenarcˇicˇ
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Introduction
A significant proportion of past and present research on peptidases was/is dedicated to the interactions between enzymes, substrates and modifiers, many of which have a direct bearing to human health. Any study of the efficacy of modifiers aimed at modulating the activity of peptidases begins in vitro, for practical reasons using synthetic substrates, which after hydrolysis of a susceptible peptide bond produce a measurable signal proportional to the concentration of hydrolyzed substrate. Enzyme kinetics provides the tools to accomplish this task, which aims at elucidating the underlying kinetic mechanisms of action. This information is necessary when formulating hypotheses on the mechanisms of action of the peptidases with naturally occurring substrates and modifiers, as well with synthetic or semisynthetic modifiers intended to be used as drugs. The kinetic tools for characterizing substrate turnover by peptidase and interactions with inhibitors and activators are dispersed between numerous specialized publications. Often, important kinetic methods are part of studies whose emphasis is placed on the biological properties of the enzymes and the ‘technical’ part is overlooked. In other instances, kinetic theories are published in journals with predominantly theoretical character and are overlooked as well. Particular methods that are not treated in specialized books can be found in the specific literature. Yet, finding these methods and putting them to work is a responsibility left to the end user. In this chapter, enzyme kinetic concepts relevant to peptidases will be discussed, while general theories can be found in excellent books dedicated to this topic (Cornish-Bowden 2004; Fersht 1977; Segel 1975). A. Baici (*) Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland e-mail: [email protected] M. Novinec • B. Lenarcˇicˇ Department of Chemistry and Biochemistry, University of Ljubljana, 1000 Ljubljana, Slovenia e-mail: [email protected]; [email protected] K. Brix and W. Sto¨cker (eds.), Proteases: Structure and Function, DOI 10.1007/978-3-7091-0885-7_2, © Springer-Verlag Wien 2013
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Symbols, Nomenclature, Conventions and Software Used
In the interest of unambiguous communication in research and teaching, especially in reporting results for publication, a consistent use of nomenclature and symbols in enzyme kinetics is highly recommended. We follow here the recommendations of the Nomenclature Committee of the International Union of Biochemistry (International Union of Biochemistry 1979, 1982). Recommended and other symbols are summarized in Table 2.1. GraphPad Prism version 5.04 for Windows, GraphPad Software (San Diego, California, USA) was used for regression analysis and graphical representations. Numerical simulations of time-dependent processes were performed with Matlab® and Simulink® (Math
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