_____________________________________________________ __ Kinetic studies of serine protease inhibitors in 'active barrier' model systems _____________________________________________________ __ Bachelor thesis by Cecilia Ålander Supervisor: Gunnar Johansson Subject specialist: Michael Widersten Examiner: Helena Grennberg Department of chemistry - BMC Biochemistry Abstract The aim of this project was to design model systems based on gelatine gel for the simulation of serine protease activity of the digestive enzymes trypsin and, briefly, chymotrypsin. Two different inhibitors, antipain and leupeptin, of the enzymes were incorporated into the models to see what kind of effect they would have on the applied enzymes. The yielded results were ambiguous for the inhibitory effect of leupeptin. The results for the antipain-affected enzyme experiments were positive showing a consistent inhibition of trypsin. It can be concluded that these models require more testing before they will be able to be directly applicable to reality. 2 TABLE OF CONTENTS 1. INTRODUCTION……………………………………………………….…...….........4 1.1. Aim........................................................................................................................ ....4 1.2. Enzyme kinetics......................................................................................................4 1.2.1. Serine proteases...............................................................................................5 1.3. The kinetics of enzyme inhibitors…………………………………………........6 1.2.1. Serine protease inhibitors...............................................................................7 1.4. Measuring kinetics…………………………………………………….……….....8 1.5. Gelatine as a model system..................................................................................8 2. MATERIALS AND METHODS……………………………………………...........9 2.1. Kinetic experiments in Petri dishes…………………………………….......….9 2.2. Kinetic experiments in 96 well Plates ……………………………………......10 2.2.1. Trypsin activity………………………………………………..….….................11 3. RESULTS ……………………………………………...............................................12 3.1 Kinetic experiments on Petri dishes. …………………………………............12 3.2. Kinetic experiments on 96 well plates.............................................................14 4. DISCUSSION…………………………………………….........................................19 4.1 Kinetic experiments on Petri dishes…………………………………..............19 4.2. Kinetic experiments on 96 well plates.............................................................20 5. REFERENCES…………………………………………………………….................22 5.1 Literature………………………….........................................................................22 5.2 Pictures………………………….............................................................................22 3 1. Introduction 1.1. Aim Proteases are enzymes with the ability to degrade or modify polypeptide structures and many are part of the human metabolism. The aim of this study is to create and test gelatine gel-based models used for the simulation of so called 'active barriers'. The models are tested by investigating how the activity of certain proteases is affected by inhibitors suspended in gelatine gel. The target enzymes of the experiments are chymotrypsin and, mainly, trypsin and the inhibitors are antipain and leupeptin. The study can be divided into two parts where the first involves enzyme activity measurements on a simple one-layered model, in which both the enzyme substrate and a relevant inhibitor is present. The second part describes experiments performed on a two-layered gel-based model system where substrate and inhibitor are separated into one layer each. 1.2. Enzyme kinetics The study of chemical reactions catalyzed by enzymes, by measuring their rate, is often generally referred to as enzyme kinetics. Kinetic analysis of reactions catalyzed by enzymes is a powerful tool used for determining the mechanism and the catalytically active parts of an enzyme. The rate of a reaction is the speed at which reactants are converted into products. The reactant of interest in enzyme kinetics is, of course, the substrate of the enzyme. Equation (1), first described by Adrian John Brown, is often used to explain the chemical reaction between the enzyme E and its substrate S (Brown, 1902). (1) The formation of the complex ES is a reversible process with a first-order rate constant for ES formation, k1 and for dissociation, k-1. Though the reaction looks simple in its design most enzymatically catalyzed reactions involve a series of chemical transformations of the substrate before it reaches its product form. These series of reactions usually include one rate determining step and can thus collectively be described by a single first-order rate constant kcat. When conducting kinetic experiments, certain conditions are usually met in order to simplify the mathematical models behind reaction mechanisms. This study uses the steady-state approximation of enzyme kinetics, which is a model widely used when studying enzyme kinetics. When an enzymatic reaction is in its steady-state, the rate of formation of the ES complex is equal to its rate of formation of product P and free enzyme. This state is achieved after rapid formation of the ES complex is balanced out by the formation of products. The initial reaction velocity v0, or simply v, of product formation (found very early in the course of the reaction) is experimentally shown to act linearly as a function of time. In this initial phase of the chemical reaction described by Equation (1) 4 only the ES complex is the only accumulating intermediate. The total enzyme concentration can thus be assumed to be as shown in Equation (2), (2) where [E]f is the concentration of free enzyme. The ES complex will accumulate until its formation is balanced out by the formation of product, since enzyme concentration [E] is very limited. The concentration of substrate [S] is in great excess compared to the catalytical enzyme concentration and is approximated to be constant during rate measurements (Copeland, 2000). The widely used Michaelis-Menten equation in Equation (3) is largely derived from the steady-state approximation and is the central equation for kinetic studies of single-substrate enzyme reactions. (3) The constant v is the initial rate of the reaction and Vmax is the maximum reaction velocity that can be achieved from saturation of the substrate concentration (see Figure 1). Km is often called the Michaelis constant in literature and is the substrate concentration, at which the initial rate velocity is half of Vmax. The Michaelis constant is often used as a measure for a substrates affinity for the enzyme, where a low Km signifies a high affinity since Vmax is quickly achieved in that system. Figure 1. A schematic picture of a diagram showing a typical 'saturation curve' with Km and Vmax marked out. 1.2.1 Serine proteases This group of enzymes specialize in degrading proteins by hydrolyzing the peptide bonds in the polypeptide chains. Many enzymes of this group are part of our metabolism where they degrade the large protein complexes into smaller peptides. Chymotrypsin and trypsin, which are studied in this project are serine proteases synthesized in the pancreas of humans and other mammals and are part of food digestion. Serine proteases are usually simple 5 enzymes with a single active site per molecule, for example chymotrypsin and have this name because their active site commonly includes a reactive serine residue. This single serine residue have proven to be crucial for the catalytic function of many proteases. By introducing an irreversible inhibitor to the enzymes that bound to the oxygen atom on serine, the activity of the enzymes was ruined (Neitzel, 2010). Chymotrypsin is a serine protease that hydrolyzes the C-terminal peptide bond of hydrophobic amino acid residues like phenylalanine, tyrosine, leucine and tryptophan. It has also had reported a secondary hydrolysis of other amino acids residues like serine, methionine, alanine, isoleucine, threonine, histidine, glycine and valine (Burell, 1993). Trypsin is a protease with a histidine and a serine residue in its active site that, similarly to chymotrypsin, cleaves peptide bonds on the C-terminal side of amino acid residues. It is more selective in its cleavage sites than chymotrypsin, only hydrolysing the peptide bonds of lysine and arginine residues (Walsh, 1970). 1.3. The kinetics of enzyme inhibitors Inhibition of an enzyme can occur in several different ways. To begin with, the inhibitors examined in this experiment are reversible inhibitors meaning that they are able to dissociate from the enzyme. It is important to not forget that some inhibitors bind irreversibly to an enzyme, however they will not be discussed in this study. Besides reversible or irreversible binding, the inhibitor can interact with an enzyme in three main forms: competitive inhibition, non-competitive inhibition and uncompetitive inhibition. This experiment focuses on competitive inhibitors which are inhibitors that only bind to the free enzyme and not to the ES complex (see Figure 2). With this kind of inhibition as long as not all free enzyme molecules are bound to the inhibitor the same maximum velocity of product formation Vmax. There will however be a competition between substrate and inhibitor for free enzyme molecules, which will increase the concentration of substrate needed to reach maximum and half maximum velocity. Km will, in other words increase