enzyme catalysis serine proteases · enzyme catalysis michaelismenten kinetic in 1913 michaelis and...

9/20/2016 Enzyme catalysis, kinetics, and allostery

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Enzyme Catalysis Serine Proteases1. The role of proteases in the cell

Proteases are proteins that cleave, or cut, or degrade other proteins by hydrolyzing peptidebonds. This may sound like an uninteresting topic, but protein degradation plays a major rolein cellular processes. Proteases are involved in cellular control mechanisms:

removal of old and unused proteins. This is an essential part of the turnoverpathway of all proteins in the cells and affects cell growth.

degradation of proteins and peptides for nutritional purposes

defense mechanisms against intrusive proteins and peptides

control of protein activity

2. Classification of proteases

The proteases are globular, water soluble proteins that function as enzymes. They catalyzethe hydrolyses of peptide bonds in proteins. Being enzymes, proteases can be characterizedby their substrate affinity and the catalytic rate of the reaction. Using proteases to study theeffects of single amino acid substitutions (mutations) on catalytic rate and substrate affinitydemonstrated that these two properties are linked and that this linkage can be explained byanalyzing the conformation of the catalytic or active site of the enzyme. This analysisshowed that four major functional groups are found in the catalytic site of proteases andbased on this functional groups, 4 families of proteases have been defined:

Serine proteases

Cystein proteases

Aspartate proteases

Metallo proteases

This classification uses the functional group within the enzyme, and does not relate to thesubstrate specificity of the proteases themselves. The name of the protease family refers to anamino acid (e.g. aspartate) or a metal as coenzyme at the active site of the enzyme.

3. Enzyme catalysis MichaelisMenten Kinetic

In 1913 Michaelis and Menten described the reaction rate and specificity for a simple onesite reaction. Here is a simplified representation of their theory. Michaelis and Mentendivided the process of the conversion of a substrate S into a product P into two steps asshown:

Km kcat E + S <=> ES <=> E + PThe first reaction step describes the binding of the substrate to the enzyme (catalyst) and theconstant Km corresponds to the dissociation constant of the equilibrium under conditionswhere the product formation is very slow compared to the dissociation process of the



substrate. Km equals the substrate concentration at half maximal reaction rate Vmax/2. In thiscase Km is a good apporoximation for the dissociation constant and thus describes the affinityof the substrate for the enzyme. For more complex reactions the constant reflects thedissociation equilibrium of all substrates bound to the enzyme.

The second reaction step describes the catalytic rate or the rate of product formation andreferred to as the turnover number kcat. The turnover rate is defined as the maximal numberof product P per active site per unit time.

The MichaelisMenten kinetic is valid only under saturation conditions, i.e., when theconcentration of substrate S is much larger than the enzyme concentration. The maximalreaction rate Vmax describes a steadystate equilibrium of the reaction catalyzed by theenzyme. The steadystate equilibrium is an important concept in biochemistry because manyenzyme catalyzed reactions run at saturation and the product often is removed from thereaction site so as to render the reaction irreversible.

The saturation conditions of an enzymatic catalysis, where the substrate concentrationexceeds the enzyme concentration, for all practical purposes, means that the MichaelisMenten kinetic is valid only for initial rates because with time the substrate gets depleted andwe no longer deal with an excess of substrate over enzyme. In addition, product P can affectthe reaction, and this is indeed a major mechanism in the control of biochemical reactions.The product can often bind to the enzyme and at a site other than the active site influence itsactivity. This is called feedback control, which can be positive or negative feed back,stimulate or inhibit the formation of P.

To complete this brief description of enzyme kinetics, we define the substrate specificity. Theratio kcat / Km defines a measure of the catalytic efficiency of an enzymesubstrate pair. Itrefers to the properties and reactions of free enzyme and free substrate. The theoretical limitfor kcat / Km is set by the rate constant of the initial complex formation (ES) and cannot befaster than the diffusion controlled interaction of substrate and enzyme. The specificity of anenzyme is therefore a measure of the specificity of an enzyme for competing substrates or ofcompeting enzymes for a single substrate. Note that specificity here should not be confusedwith the use of the term 'specificity' instead of 'affinity', as in 'specificity pocket' discussedbelow.

4. Activation energy

The rate of an enzymatic reaction is strictly dependent on the activation energy of thecatalyzed reaction. Enzymes catalyze chemical reactions by lowering the activation energy ofthe reaction. This is achieved by either the binding of two substrates in an orientation optimalfor the reaction to occur, or by using differential binding energies for transition state of S* ascompared to the initial bound state of S (binds S* more tightly than S).

5. Chymotrypsin super family of proteases

Chymotrypsin, trypsin, and elastase are three members of the super family of chymotrypsinproteases. They all are serine proteases because the amino acid residue at the catalytic siteresponsible for the transition state stabilization is a serine. These three proteins have differentoverall structures but identical active site conformations. The active site of a serine proteasecan be divided into four essential structural features required for the catalytic action of serineproteases: Structural element of active site Function___________________________________________________________________



The main chain substrate binding unspecific binding of polypeptide segment

Specificity pocket semispecific binding of side chains,sequence specificity

Oxyanion whole stabilizes S* over S in enzyme

The catalytic triad forms tetrahedral intermediate (transitionstate; stabilizes S* over S); hydrolyzespeptide bond

6. The reaction mechanism of chymotrypsin

The protease chymotrypsin is synthesized as an inactive precursor protein, called Chymotrypsinogen, consisting of 245 amino acids. The enzyme is activated by the proteolyticremoval of two dipeptides at positions 1415 and 147148. The active protein consists ofthree polypeptide chains covalently linked through SS bonds, and noncovalently throughhydrogen bond formation and nonpolar interactions. Therefore, the native complex, althoughcleaved into three polypeptides, does not fall apart. The protein consists of 2 domains that areapproximately equal, containing ~120 amino acids each and are build from an antiparallelbbarrel.

Fig. Domain structure of a chymotrypsin type serine protease with the catalytictriad at the domain interface (Asp102 and His57 in domain 1 and Ser195 indomain 2).

Protien database accession number for serine proteases are 1PD0 or 1AQ7 for trypsin, 1GMD forchymotrypsin, 1LVY for elastase, and 1CSE for subtilisin (+inhibitor).

The six step reaction mechanism of chymotrypsin:

1. substrate attaches to enzyme at the main chain binding site and specificitypocket with the scissile (peptide) bond exposed to the triad (ES formation)

2. covalent bond formation between C=O and S195 hydroxyl (nucleophilicattack), tetrahedral intermediate as transition state, oxyanion hole stabilization,H57 binds the released H+ from S195 (ES*)

3. acylenzyme intermediate and peptide bond hydrolysis, peptide with new Nterm released from enzyme taking H from H57 (originally S195; see step 2)(EP*)

4. a water molecule placed next to H57 and acylenzyme intermediate at S195(EP*)



5. the water molecule initiates a nucleophilic attack and undergoes a reactionwith one H attaching to H57 and OH to acylenzyme intermediate formingagain a tetrahedral transition state of the new Cterminal peptide fragment (EP*)

6. peptide released while H57 donates H (from water molecule) to S195 (E+P)

Fig. Peptide backbone with scissile bond between residue n1 and n. Residue n1 fits intospecificity pocket of enzyme.

7. Specificity pocket

Proteases have preferential cleavage sites in the sequence of a protein substrate. Thespecificity pocket provides a small binding pocket consisting of 3 amino acid residues thatdetermine the local polarity and electrostatic potential profile for the interaction of residue n1 on the substrate on the Nterminal side of the scissile bond. For the chymotrypsin family ofserine proteases we find the following sequence specificities:

Table Specificity pocket substrate interaction for the Chymotrypsin Superfamilyserine protease chymotrypsin trypsin elastase______________________________________________________________Rn1 bulky, aromatic + charged small, noncharged

Note: residue at position n can not be a proline because of its closed, inflexible ring structure.

The 3D folds of the three proteases are very similar, although there is no substantialsequence similarity. Another serine protease family, subtilisin family of serine proteases, areproducts of bacilli species. Their overall native fold is very different from that of thechymotrypsin type proteases, but the catalytic site conformation is identical. The 3D fold ofsubtilisin has an a/b motif (instead of the b barrel motif of chymotrypsin domains) with fiveparallel bstrands surrounded by 4 ahelices.

The comparison of the two families of serine proteases tells us two different things. First, ithas been reasoned to be an example of convergent evolution, where the formation of acatalytic site has evolved twice, with each protease family exhibiting a different overall 3Dstructure. Second, the differences in the 3D structure gives us an idea of the differentcellular locations of the corresponding protein families: the catalytic site of an enzyme isconserved over evolutionary time, while the overall structure is conserved to providestructural stability for optimal activity of the protein in any given environment. We need onlyunderstand that very different sequences can provide similar 3D structures because watersolubility depends only on the distribution of hydrophilic and hydrophobic residues, but noton other chemical properties. Overall structural features thus reflect the location of theprotein, if it is located intracellular, extracellular, if it is cell membrane protein, or if it isresistant to temperature changes or sensitive to proton or calcium concentrations.



8. Protease Inhibitors

Cellular control of proteases is carried out by protease inhibitors. These are small peptides orproteins that can bind to the active site of the protease (competitive inhibitor) but which arenot hydrolyzed, thereby blocking the access for substrate proteins. One example of a proteaseinhibitor is the bovine pancreatic trypsin inhibitor (BPTI), a small protein of 58 amino acids.Its structure has been determined by Xray crystallography and the protein has been widelyused for folding studies of proteins (see section 2.7). BPTI binds to trypsin through hydrogenbonding forming a tightly packed interface between inhibitor and enzyme. The MichaelisMenten constant of BPTI binding Km = 1013M. The lysine at position 15 binds to thespecificity pocket followed by an alanine. The reaction is blocked at the formation of thetransition state intermediate.

Allostery and Cooperativity Hemoglobin

1. Introduction

Most enzymes are protein complexes. Single protein enzymes, such as serine proteases, arerare. The discussion of the structurefunction relationship of the oxygen binding myoglobinand hemoglobin provides an insight to the advantage of polypeptide association into largerfunctional complexes. Protein complexes provide structural and functional variabilitiesthrough combinatorial effects of subunits that cannot be achieved by single polypeptides (ofcourse they could be large, multidomain proteins, where each domain adopts the function ofa subunit). The single most important functional aspect, however, affects the regulation of theprotein complex. Oligomerization enables cooperativity between multiple binding sites, aproperty referred to as allostery or allosteric regulation of enzymes (yet again, singlepolypeptide, multidomain proteins show allosteric behavior).

Protein complexes are often referred to as oligomeric proteins. Oligomeric proteins arecomposed of subunits, with the subunits being individual polypeptide chains. Proteincomplexes have defined quaternary structural symmetries, if they are homooligomericprotein complexes where all subunits are identical. The quaternary symmetry are pseudosymmetries if the complex is a heterooligomer. Homo and heterooligomeric compositionsallow for an additional regulatory hierarchy at the cellular, tissue, or organism level, wheredifferent combinations of subunits of heteromeric complexes, due to cell specific geneexpression, often show minute, yet important differences in enzyme activity.

Two protein systems will be discussed in detail to elucidate the structurefunctionrelationship in oligomeric proteins. The first system is the globin protein family. Members ofthis family are the myoglobin, a single polypeptide enzyme, and hemoglobin, a tetramericprotein complex with a2b2 stoichiometry (where the Greek letters denote subunit types, notsecondary structure). Both globins are oxygen binding proteins. Myoglobin has one bindingsite, whereas hemoglobin has four binding sites, one per subunit. Myoglobin and hemoglobindiffer in their regulatory capacity of binding O2. The four binding sites in hemoglobin showpositive cooperative binding, meaning that after the first binding site is occupied by an O2molecule, the 3 remaining sites exhibit a largely increased affinity for O2 molecules.

The second protein system is the nicotinic acetylcholine receptor,ornAChR, in postsynapticmembranes of skeletal muscle and neurons (see section 2.3). This receptor is a heteromericprotein complex of five integral membrane proteins with a subunit stoichiometry of a2bgd.The five subunits are arranged in a circular fashion around a central hole that provides an ionpathway across the cell membrane. The pentameric complex has a five fold pseudo



symmetry because its subunits are not identical. The receptor complex binds two moleculesof acetylcholine. Acetylcholine binding induces the opening of the channel therebytransmitting a chemical signal (neurotransmitter binding) into an electrical signal (ion flux).The coupling of the ligand binding site and the channel structure (active site) is an allostericeffect because the binding of the ligand causes a structural change at a distant place on thereceptor unit.

Both oligomeric protein systems, although they have completely different functions, use asimilar mechanism that makes a hemoglobin a cooperative (crosstalk between identical units)unit and the nAChR an allosteric ion channel (crosstalk between different units). Thedistance between two heme binding sites in hemoglobin is about 2.5nm and the distancebetween the acetylcholine binding site and the channel gate, the structure in the complex thatopens and closes the ion pathway across the membrane, is 2.5nm as well.

2. Structure of Myoglobin and Hemoglobin

Myoglobin is a small globular protein that binds O2 in muscle tissue. Its 'enzyme' activity isnot the catalysis of a reaction, but to increase the water solubility of oxygen and to facilitateits transport to the muscle cell. Its structure was the first to be solved in its entirety by Xraycrystallography (Kendall et al., 1961). It contains 153 amino acids and is folded into 8 ahelices, labeled A through H, connected through short loop structures. The ahelix motif ofmyoglobin is also called the globin fold. The O2 binding site consists of a protoporphyrin IXring with a centrally coordinated Fe(II) forming the heme group which is noncovalentlyembedded in the globin fold between helices E and F. The central Fe(II) coordinates with thefour N in the protoporphyrin IX structure. The fifth coordination site is occupied by His93amine (helix F). The sixth coordination place is free and accessible for an O2 molecule.Because of its asymmetric coordination state in the oxygen free state, the Fe is slightlypositioned off the protoporphyrin ring plane. The binding of an oxygen molecule pulls theFe(II) into the plane of the heme. The His93 is pulled along with the Fe(II) and as aconsequence displaces the Fhelix at one end by about 0.1nm.

Comparing the amino acid sequences of related globins for the helixhelix contacts shows nosimilarity. Obviously different amino acids can be replaced without disrupting the over allglobin fold. The hemepocket, however, is an evolutionary conserved structure. Mutations inthe globin fold that do not affect the conformation of the hemepocket are accepted, even ifthey might slightly affect relative positions between helices or loop structures.

Hemoglobin is a tetrameric protein complex composed of two alpha and two beta subunitswith an overall subunit stoichiometry of a2b2. The hemoglobin tetramer is a spheroidalprotein complex with the dimension 6.4 x 5.5 x 5.0 nm. The tetramer has a pseudoD2symmetry and the association between subunits is stabilized by hydrophobic interactions aswell as hydrogen bonds. The overall sequences and structures of the hemoglobin subunits arehomologous to myoglobin. Their arrangement into a tetramer, built from the association oftwo ab dimers has important consequences with respect to oxygen binding affinity andregulation. Hemoglobin contains 4 heme groups, all of which are bound to each individualsubunits as shown for myoglobin. The binding of the first O2 to any of the hemes induces ashift in the corresponding Fhelix. This structural change within a subunit is sensed by theneighboring subunits in a way that the binding of O2 to their hemes is favored as comparedto the initial conformational situation. Thus, the binding of a first O2 induces a decrease inthe binding energy for subsequent O2 binding in the other three subunits.

This mechanism is called cooperative effect.

3. Hill coefficient a measure of cooperativity



Both myoglobin and hemoglobin bind O2 and transport it to their target. Why is one type ofglobin not enough? Looking at the physiology of transporting oxygen to our muscle cells andkinetically analyzing the effects of cooperativity and noncooperativity of O2 binding, we canobserve a physiological system that provides both a high efficiency and tight control over O2transport to the electron transport systems in muscle cells that convert the high energycontent of O2 bond electrons upon oxidation into metabolic energy. To understandcooperativity of O2 binding we have to define the fractional saturation, YO2, of globin withO2 molecules. The fractional saturation can experimentally be determined by measuring thepartial oxygen pressure, pO2, in solution. The fractional saturation and the partial oxygenpressure are related as shown in this equation:

p(O2)n Y(O2) = p(50)n +p(O2)n

p(50) is defined as partial pressure of O2 to upon 50% occupation of all heme binding sitesby ligands. The cooperativity factor is indicated by the factor n; for hemoglobin n = 3; formyoglobin n = 1.

Fig. Fractional saturation of Myoglobin (Mb) and Hemoglobin (Hb)

The different behavior of oxygen binding of myoglobin and hemoglobin can be summarized asfollows. Myoglobin binds O2 under conditions where hemoglobin releases it. This is below 20Torr in muscle tissue. At this pressure, hemoglobin releases almost all of its oxygen andmyoglobin binds the freed oxygen at over 90%. The hyperbolic curve of Mb binding is typicalfor noncooperative processes, while the sigmoidal curve of Hb binding is typical forcooperativity.

A measure of cooperativity was defined by Hill, when plotting the partial pressure against thefractional saturation of the globin. The steepness of the slope (at 50% saturation) in a doublelogarithmic graph has been defined as Hillcoefficient. A Hill coefficient of 1 indicates noncooperativity as found for myoglobin (only one binding site, obviously there is no interactionbetween two myoglobin molecules). A Hill coefficient larger than one indicates positivecooperativity. For hemoglobin the value is ~3. This has been interpreted that upon binding ofthe first O2 the other three bind almost immediately.

4. Mechanism of cooperativity of O2 binding



The structural changes in Hb subunits can be analyzed and a kinetic model for thosestructural changes has been described. In this model, the quaternary structure of hemoglobinwith no molecular oxygen bound, deoxyHb, is designated as Tstate and the fully oxygenatedform, oxyHb, as Rstate. Similarly the subunit forms are designated t and r states. It isimportant to understand that the occupancy state of one heme cannot be sensed by an otherheme by direct electronic interaction. They are too far away (>2.5nm). The interaction is amechanical one, transmitted through the conformational change induced by the binding ofone O2 to a heme and the consequent displacement of the FHelix. This helix displacementbrings upon a change in the quaternary structure such that the dimer (a1b1) rotates 15°relative to the a2b2 dimer. The dimers a1b1 and a2b2 relative subunit orientation isunaffected.

Fig. Quaternary conformational change in hemoglobin (Fermi, p.169)

It is the movement of the Fe(II) towards the heme plane that triggers the T to Rconformational change. The relative displacement between subunits has only two stableconformations, i.e., the T and the R state. Intermediate positions are energeticallyunfavorable, therefore the change in quaternary structure provides two conformationalendstates, one with a low O2 affinity, the other with an increased affinity to the heme groups.These two states are stabilized by two energetically equivalent sets of hydrogen bonds at thesubunit interfaces. After the binding of the first oxygen, all subunits are converted from the tto the r state whether or not they bind a ligand.

Fig. The hydrogen bond contacts at the a1b 2 interface (Fermi, p.180)



In general, cooperative interactions occur when ligands bind to proteins or proteincomplexes. If the binding of ligands is regulated by different ligands at sites other than theheme, then we call this allosteric interaction. Ligands function as effectors or modulators atdifferent sites in the protein. If the ligands are identical, this is known as homotropic effect, ifthey are different, it is known as heterotropic effect. Molecular oxygen is a positive,homotropic effector for hemoglobin. The protein also exhibits heterotropic effects whichinduce mostly negative cooperativity, i.e., they decrease the affinity of O2 to hemoglobin. Forexample the generation of CO2 in the muscle and its conversion into bicarbonate releasesprotons. These protons bind to Hb and reduce its affinity for O2 by stabilizing the T state.This effect is known as Bohr Effect. In addition, CO2 binds reversibly to hemoglobin at itsNH2 terminal end to form carbamates (RNHCOO). CO2 binds better to the Nterminal endof deoxyHb, and once bound, stabilize the T state.

Fig. pH dependence of oxygen binding to hemoglobin

Oxygenation changes the electronic state of the Feheme complex, a change that can bemeasured spectroscopically as a slight shift from purple to red. O2 binding is competed byCO, NO, and H2S. In hemoglobin this binding is stronger than that of O2 causing death byquickly binding to the deoxygenated hemoglobins.

5. The symmetric model of allostery in protein complexes

The symmetry model of allosterism describes the cooperative binding of ligands to anenzyme complex. It was developed by Jaques Monod, Jeffery Wieman and JeanPierreChangeux. It is therefore also called the MWC model of allosterism and is defined by thefollowing rules:

An allosteric protein is an oligomer where all subunits are symmetricallyrelated.

Each subunit exists in two (or more) conformations t and r, they are inequilibrium whether ligands are bound or not.



The ligand can bind to a subunit in either conformation, the conformationalchange alters ligand affinity.

The molecular symmetry is conserved during conformational change. Subunitstherefore change in a concerted manner, there are no oligomers containing both tand r states.

enzyme catalysis serine proteases · enzyme catalysis michaelismenten kinetic in 1913 michaelis and...

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