Computational Biology 1

Transcription

1 Computational Biology 1 Protein Function & nzyme inetics Guna Rajagopal, Bioinformatics Institute, guna@bii.a-star.edu.sg References : Molecular Biology of the Cell, 4 th d. Alberts et. al. Pg

2 Levels of Protein tructure econdary structure elements combine to form tertiary structure Quaternary structure occurs in multienzyme complexes Many proteins are active only as homodimers, homotetramers, etc.

3 Protein Domains A sub-structure produced by any part of a PP chain that can fold independently into a compact, stable structure. It is a modular unit from which much larger proteins are constructed. Central core of a domain can be constructed from alpha helices and beta sheets in various combinations.

4 A protein formed from 4 different domains rc protein. Two of the domains form a protein kinase enzyme while the H2 and H3 domains perform regulatory functions.

5 ignature sequences to find protein domains Multi-domain proteins believed to have originated when the DNA sequences encoding each domain accidentally became joined, creating a new gene. Many proteins show signs of having evolved by the joining of pre-existing domains in new combinations via domain shuffling. equence homology searches can identify close relatives among proteins. H2 domain

6 Protein Families Once a protein had evolved that folded up into a stable conformation with useful properties, its structure could be modified slightly during evolution to enable it to perform new functions.. Members of the same protein family have AA sequence and 3D conformation that closely resembles all of the other family members.

7 Importance of intermolecular interfaces in Quaternary structures A number of genetic diseases (e.g. sickle-cell cell anemia) originate from hydrophobic surfaces that are inappropriately created as a result t of a mutation. The mutation of glutamate (hydrophilic) to valine (hydrophobic) at the surface of the beta unit of hemaglobin creates a hydrophobic patch that causes hemoglobin tetramers to polymerize into long fibrils.

8 elf-assembly

9 Protein Function

10 Overview of Thermodynamics

11 A Basic Glossary for Thermodynamics Heat (Q)- That quantity of energy exchanged across the boundary of two systems that results in a change in temperature. Work (W) - Work is force times distance. Roughly heat is motion on the microscopic scale and work is motion on the macroscopic scale. Both have units of energy. nergy () - nergy is the capacity for doing work. In thermodynamics this is the internal energy. nthalpy (H) - The heat content of a system. ( H = PV) ntropy () - A measure of the unavailable energy in a closed system. Also a measure of disorder. Free nergy (G) - The tendency of a system to change if a channel to do so is available. (G G = H T)

12 Disorder and the relationship to entropy Disorder and entropy are intimately related. Ludwig Boltzmann c Why doesn t the gas spontaneously reappear back in the box?

13 One Three Laws of Thermodynamics 0 th Law- Two systems that are in thermal equilibrium with a third system are in equilibrium with each other. 1 st Law - The total energy of system plus surrounding is conserved. 2 nd Law - The total entropy of the system plus surroundings never decreases. 3 rd Law - The entropy of all pure, perfect crystals at absolute zero (elvin) is zero.

14 Gibbs Free nergy The Gibbs Free nergy, G, is a state function, defined as: G H T At constant temperature and pressure, G is G = H T For spontaneous events, we have tot 0 G 0 Josiah Willard Gibbs ( ) 1903)

15 Chemical Reactions What makes a reaction spontaneous? nthalpy ntropy H 298 (A) H 298 (B) H 298 (C) Free energy H= H(B)H(C)-H(A) >0 G = H T What makes a reaction actually happen? Molecular Detail, Transport and inetics!

16 Catalytic Action

17 inetic Barriers

18 The Gibbs Free nergy is a direct measure of spontaneity G = H T sums up, in a way, the competition between energy considerations and configurational barriers. A process is spontaneous if Thus, if o G G < 0 H < 0 the process is exothermic (downhill) > 0 the process is increases disorder ntalphy, H dominates spontaneity at low temperatures ntropy, dominates spontaneity at high temperatures

19 Thermodynamics of protein folding ince G = H T H Is negative due to more favorable hydrophobic interactions on the folded state Is negative as the folded state is more ordered (so has lower entropy). At finite temperature, this balance is very delicate and the free energy difference between the folded and unfolded state is about 15 kcal/mole (approx 3 hydrogen bond energies!)

20 Proteins as nzymes

21 Why is enzyme kinetics important? Vital to understand detailed mechanisms to provide insight for: drug discovery, large scale industrial synthesis of useful chemicals, fundamental understanding of biochemistry of cells and organisms, We need to determine how reaction rates change in the presence of nzymes with changes in conditions such as concentration of substrates, products, inhibitors, regulatory ligands, environment,

22 Mechanism of nzyme Action nzymes are proteins that first bind tightly to specific molecules called substrates, and then catalyze the making and breaking of covalent bonds in these molecules. At the active site of an enzyme, the amino acid side chains of the folded protein are precisely positioned so that they favor the formation of the high-energy transition states that the substrates must pass through to react.

23 Binding ite Folded protein contains a crevice or cavity on the protein surface. This crevice contains a set of AA side chains disposed in such a way that they can make non-covalent bonds only with certain ligands. camp

24 The biological function of a protein depends on the detailed chemical properties of its surface and how it binds to other molecules, calledc ligands.

25 nzyme inetics

26 Overview Many different mechanisms for enzymatic function k1 k 1 k cat P Michaelis-Menten Menten I β I k1 k 1 k cat Competetive Inhibition P = nzyme = ubstrate P = Product 1 k1 k 1 1 kcat iso-ordered Ping-Pong Pong 2 β 2 kcat P 1 P 2 I = Inhibitor = enzyme/substrate complex, etc.

27 For all these reaction types there are a fairly algorithmic way of solving for their rate laws.. In all cases this involves: 1) Writing them down as elementary reaction steps 2) Making approximations: a) stationary state approximation b) rapid equilibrium approximation c) conserved total enzyme d) large but not too large concentration of s e) small but not too small concentration of f) we are interested in only initial rates

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29 Michaelis-Menten Menten Mechanism k1 k 1 k cat Basic idea (will give a detailed mathematical treatment later) olved using the stationary-state state approximation: v 0 t = k d[ ] dt cat [ ] = [ ] [ ] = k 1 [ ][ ] ( k 1 k cat )[ ] = 0 P nzymes, [], are present at about M. ubstrates, [], present at more like micromolar to millimolar k 1 ( t [ ] = [ ])[ ] k 1 t k k 1 cat ( k 1 = k t M cat )[ ] = 0 v = velocity 0

30 Vmax =t cat [] The rate of product production (i.e. the velocity of the reaction) for a Michaelis-Menten Menten mechanism is: v 0 = k [ ] cat k cat is a measure of the intrinsic activity of the enzyme. = k cat M t v 0

31 Lineweaver and Burk Plots Lineweaver-Burk plots are linear for Michaelis-Menten Menten enzymes. Note that they give a measure of m and V max. These rates are also called turn-over numbers since they are a measure of how many substrate molecules the enzyme can process per second working at maximum rate! However, the overall rate of the production of product from substrate is lower due to the Collision probability term.

32 nzymes can be affected by binding of non-substrate molecules. Inhibition can occur through numerous routes. I β I k1 k 1 k cat Competitive Inhibition P I β I k1 k 1 β I k cat P Non-competitive Inhibition

33 The effect of a competitive inhibitor is to remove some enzyme from f the pool available to interact with. I β I t I k1 k [ ] = 1 = [ ][ ]/[ ] = [ ][ I]/[ I] k cat = [ ] [ I] [ ] = [ ] = [ I] [ ] t s I [ ] [ ] P t[ ] [ I] (1 ) [ ] Competitive Inhibition [ I] [ ] [ ] [ ] The apparent effect is to lower the apparent binding constant of the enzyme for the substrate. I I

34 On the other hand for a system like this below we get: I β I t I = [ ][ ]/[ ] = [ ][ I]/[ I] k1 k 1 = I t [ ] 1 [ ] I [ ] t [ ] = [ ] I 1 I β I 1 k cat [ I] = [ ] [ I] [ I] [ ] = [ ] [ ] I I P Non-Competitive Inhibition I [ I] [ ] [ ] [ ] [ ] Which shows that the net effect of the inhibitor is to decrease the apparent max of the enzyme. V max I

35 [ ] = Uninhibited [ ] t [ ] t[ ] [ ] = [ I] (1 ) [ ] I Competitive [ ] t [ ] = [ ] I 1 Non-Competitive The effects on a Linweaver-Burk plot: I y-intercept= 1/V max slope= m /V max x-intercept= -1/ M

36 Allosteric Proteins The three-dimensional structure of many proteins has evolved so that the binding of a small ligand can induce a significant change in protein shape. Most enzymes are allosteric proteins that can exist in two (or more) conformations that differ in catalytic activity. They can be turned on or off by ligands that bind to a distinct regulatory site to stabilize either the active or inactive conformation.

37 Myoglobin and Hemoglobin help to partition oxygen into the blood stream. However, the binding of oxygen to these molecules is very different.

38 Cooperativity Fraction O2 bound Myoglobin Hemoglobin Concentration of O2 For Hemoglobin we get a very different curve for the binding isotherm when compared to myoglobin. This is not due to the fact that hemoglobin has 4 sites for binding, rather it is due to interactions among binding sites i.e. cooperative effect.

39 Protein Phosphorylation A protein s function can be regulated by the addition of a phosphate group covalently to one of its AA side chains. Addition/removal of phosphate groups from specific proteins often occurs in response to signals that specify some change in a cells state.

40 Regulation of catalytic activity of proteins xample of feedback inhibition of a single biosynthetic pathway. Here, the end product Z inhibits The first enzyme that is unique to Its synthesis and thereby controls Its own level in the cell. An example of negative regulation.