Enzymes Nearly all the reactions of the body are mediated by enzymes Enzymes are protein catalysts that increase the rate of the reactions without being changed in the overall process Catalysts for biological reactions Lower the activation energy Increase the rate of reaction Activity lost if denatured May be simple proteins May contain cofactors such as metal ions or organic (vitamins)

Enzymes Nomenclature of enzymes Enzyme has two names a. Short Recommended name b. Systematic name Recommended name End in –ase, Identifies a reacting substance sucrase – reacts sucrose lipase - reacts lipid Describes function of enzyme oxidase – catalyzes oxidation hydrolase – catalyzes hydrolysis lactate dehydrogenase, adenylate cyclase… Common names of digestion enzymes still use which don't provide any hint as pepsin and trypsin

Systematic name The international union of Biochemistry and Molecular Biology (IUBMB) developed a system for nomenclature in which enzymes are divided into 6 groups and sub classes. These names are unambiguous and informative but sometimes long and difficult to be of general use. Class Reactions catalyzed Oxidoreductoases oxidation-reduction Transferases transfer group of atoms Hydrolases hydrolysis Lyases add/remove atoms to/from a double bond Isomerases rearrange atoms Ligases combine molecules using ATP

Enzyme Action: Lock and Key Model An enzyme binds a substrate in a region called the active site Active site is a special pocket or cleft in the enzyme molecule The active site contains amino acids side chains that form a three dimensional surface complementary to the substrate Only certain substrates can fit the active site The active site binds to the substrate and form enzyme-substrate complex that will dissociate into the enzyme and product. Amino acid R groups in the active site help substrate bind

Lock and Key The active site of the unbound enzyme is complementary in shape to that of the substrate

Enzyme Action: Induced Fit Model Enzyme structure flexible, not rigid Enzyme and active site adjust shape to bind substrate Increases range of substrate specificity Shape changes also improve catalysis during reaction The enzyme changes shape upon binding substrate The active site has a shape complementary to that of the substrate only after the binding

Enzyme-Substrate Complex

Cofactors Some enzymes associate with non-protein cofactor that is needed for enzymatic activity These cofactor include metal ions (Zn, Fe) and organic molecules called coenzymes that often derivative of vitamins; NAD+, FAD, CoenzymeA.. Holoenzyme refers to the enzyme with its cofactor, Apoenzyme refers to the protein portion of the holoenzyme and it dose not show biological activity. Prosthetic group is a tightly bound enzyme that dose not dissociate from the enzyme Location of enzymes Many enzymes are located into specific organelles in the cell serve to isolate the reaction substrate or product from each other and to provide a special environment for a reaction and to organize these reactions

Turnover number and catalytic efficiency Enzyme-catalyzed reactions are highly efficient, proceeding from 103 – 108 times faster than unanalyzed reactions. The number of substrate molecules converted into product per enzyme per second is called Turnover number, typically each enzyme molecule is capable of transforming 100-1000 of substrate molecules into product per second Enzymes are highly specific, interacting with one or a few specific substrate Enzymes could be enantiomers specific

How Enzymes work The mechanism of action of enzymes can explained by two different modes Energy changes during the enzyme-catalyzed reaction, enzyme provide an energetically favorable reaction pathway different from unanalyzed reaction The active site chemically facilitates catalysis Energy Changes during the reaction - the reactant and the product are separated by energy gap or energy barrier that called free energy of activation and it equals to the energy difference between the energy of reactant and the energy of high-energy intermediate (Transition state) A  T*  B The peak of energy represents the Transition state in which the high-energy intermediate is formed during the conversion of reactant into product. because of large activation energy the rate of unanalyzed reaction is slow

Rate of Reaction -The substrate molecules should have sufficient energy to overcome energy barrier to be converted into products. only few molecules have energy to pass the energy gap  rate of reaction is determined by number of molecules that is converted into the product. the lower the free energy of activation, the more molecules have sufficient energy to pass over the transition state the faster the rate of the reaction

The enzymes don’t change the free energies of the reactants and products  don’t change the equilibrium of the reaction

Chemistry of the active site The binding of a substrate to the binding site involves a complex molecular machine chemical reactions  facilitate the conversion of substrate to product. Many factors responsible for enzyme catalytic efficiency of enzymes: Transition state stabilization: the active site often act as a flexible molecular template that binds substrate  stabilize the transition state  increase the product production Active site provide catalytic groups that enhance the probability that transition state forms, these groups participate in general acid base catalysis in which amino acids can accept or provide protons, in other enzymes catalysis may involve the transient formation of a covalent enzyme-substrate complex

Transition State

Factors Affecting Enzyme Action Different enzymes show different response to changes in substrate concentration, temperature, and pH Reaction Rate (velocity of a reaction): Reaction rate is the number of substrate molecules converted to product per unit time and is usually expressed as µmoles product formed per minute The rate of reaction (enzyme catalyzed) increases with substrate concentration until a maximal velocity (Vmax), the plateau reflects the saturation with a substrate of all available binding sites on the enzyme substrate concentration Maximum activity Reaction Rate Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant) Maximum activity reached when all of enzyme combines with substrate

substrate concentration Maximum activity Reaction Rate Vmax Vmax/2 Km Most of enzymes show hyperbolic dependence of velocity on substrate concentration

Factors Affecting Enzyme Action: Temperature Little activity at low temperature Rate increases with temperature (the velocity increased with Tem until a peak due to the increased number of molecules having sufficient energy to pass over the energy barrier and form product) Most active at optimum temperatures (usually 37°C in humans) Activity lost with denaturation at high temperatures decrease the velocity Optimum temperature Temperature Reaction Rate High Low

Factors Affecting Enzyme Action: pH pH affect the ionization of the amino acids in the active site. R groups of amino acids in the active site should have proper charge to bind with the substrate the ionization or unionization is affected by the pH Maximum activity at optimum pH Tertiary structure of enzyme is correct Most lose activity in low or high pH, extremes of pH can also lead to denaturation of the enzyme because the structure of active protein depends on the ionic character of the amino acid 3 5 7 9 11 pH Reaction Rate Optimum pH Narrow range of activity, and the pH optimum varies for different enzymes

Each enzyme has its optimum pH

E + S ES E + P V [S] Rate v = km + [S] Michaelis-Menten Model k1 k2 Michalis and Menten proposed model that accounts for most features of enzyme-catalyzed reactions. In this model the enzyme reversibly binds its substrate to form ES complex that subsequently breaks down to product E + S ES E + P k1 k2 k-1 Where E: enzyme S: substrate ES: enzyme-substrate complex P: product k1 k-1 k2 are rate constants Michaelis-Menten Equation The Michaelis-Menten Equation describes how reaction velocity varies with substrate concentration V [S] Rate v = km + [S]

Assumptions Required for Michaelis-Menten Equation E and S combine to form ES complex There is only a single substrate, the concentration of S is much greater than concentration of enzyme the amount of the substrate bound by the enzyme at any one time is small. E + S = ES rapidly reaches equilibrium: steady state assumption, concentration of ES is constant, dose not change with time the rate of formation of ES is equal to that of the breakdown of ES (to E and P) Reaction from P is irreversible only the initial reaction velocities are used in the analysis of enzyme reactions- the rate of reaction at zero time, at that time the concentration of eth product is very small  the rate of the back reaction from P to S can be ignored Michaelis-Menten Equation Vo: initial reaction velocity Vmax: maimal velocity Km: michaelis-menten constant= (k-1+K2)/K1 [S]: concentration of substrate V max[S] vo = km + [S]

Michaelis-Menten Model

Michaelis-Menten Model

Conclusions about Michaelis-Menten kinetics Characteristics of Km: The Km constant is a characteristics of an enzyme and particular substrate Km reflects the affinity of the enzyme for a particular substrate Km numerically equal to the concentration substrate at which the reaction velocity is equal to ½ Vmax. Km dose not vary with concentration of the enzyme low Km  high affinity; low [S] is needed to half-saturate the enzyme large Km  low affinity; high [S] is needed to half-saturate the enzyme

Relationship between the velocity to enzyme concentration: the rate of reaction is directly proportional to the enzyme concentration at all substrate conc. if the enzyme concentration is halved  the Vo is reduced to half Order of reaction: When [S] << Km, V (velocity of reaction) is roughly proportional to [S] the rate of the reaction is first order reaction When [S] >> Km, the V is constant and equal Vmax. The rate of reaction is independent on the [S] the reaction rate is Zero order

Double reciprocal of Michaelis-Menten equation Lineweaver-Burk Plot When V is plotted versus the [S], it is not always possible to determine the Vmax or Km from the graph because of gradual upward slope of the hyperbolic curve vo =Vmax [S] / (Km + [S]) (Michaelis-Menten Equation) Equation of straight line: y= ax + b (a = slope, b = y intercept Linear Transform: take reciprocal of each side of equation 1/vo = Km/Vmax • 1/[S] + 1/V y = a x + b

Double reciprocal of Michaelis-Menten equation Lineweaver-Burk Plot

Lineweaver-Burk Double Reciprocal Plot 1/vo = Km/Vmax • 1/[S] + 1/Vmax Lineweaver-Burk line allow us to determine the Vmax and Km simply The x intercept is -1/km The y intercept is 1/Vmax

What do we mean by inhibitors?

Enzyme Inhibition Enzyme inhibitor: any substance that can reduce the velocity of an enzyme-catalyzed reaction and cause a loss of catalytic activity Inhibitors can be reversible or irreversible. - Reversible inhibitors bind to enzymes through a non-covalent bonds  Upon dilution the EI (enzyme-inhibitor) complex dissociate and recover the enzyme activity, may be competitive or noncompetitive - Irreversible inhibition occur when the inhibited enzyme can not recover its activity by dilution. The inhibitors form a covalent bonds with the active site enzyme or destruction of the protein structure of the enzyme Irreversible Inhibitors

Competitive inhibitors This type of inhibition occurs when the inhibitors bind reversibly to the same site that the substrate normally occupy  compete the substrate for that site A competitive inhibitor Has a structure similar to substrate Occupies active site Competes with substrate for active site Has effect reversed by increasing substrate concentration

Competitive inhibitors Vmax: the effect of the competitive inhibitors is reversed by increasing the [S]. At sufficient high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of the inhibitors. The y-intersect is unchanged Km: a competitive inhibitors increases the apparent Km for a given substrate in the presence of this inhibitors , more substrate is needed reach the Vmax. The x intersect is changed indicating that the apparent Km is increased

Non-Competitive Inhibitors Inhibitor binds at a site distinct from the substrate site It may bind to free E or to ES. Once bound it will prevent P formation. And the affinity of the I to both E and Es is the same.

Noncompetitive Inhibition Non competitive inhibition occurs when the inhibitor at a site distinct from the substrate site A noncompetitive inhibitor does not have a structure like substrate. Alter the shape of enzyme and active site Substrate cannot fit altered active site It binds either to the enzyme or to the ES complex No reaction occurs Effect is not reversed by adding substrate It may bind to free E or to ES. Once bound it will prevent P formation. And the affinity of the I to both E and ES is the same.

Non-competitive inhibitors decrease the Vmax, and can not be overcome by increasing the substrate concentration Non-competitive inhibitors don’t affect the Km, the enzyme show the same Km in the absence or the presence of the inhibitors

Enzyme inhibitors could be used as drugs Different types of enzyme inhibitors I 2 I 1 I 0 NONCOMP COMP

Allosteric Enzymes and Allosteric Regulation Allosteric enzymes: are regulated by molecules called effectors (modulators) that bind non-covalently at site other than the active site. Allosteric enzymes show Sigmoidal curve and don't follow Michalaelis-menten rules Allosteric modulators affect the enzyme affinity to its substrate and /or modify the maximal catalytic activity of the enzyme Enzyme regulation is essential to coordinate the numerous metabolic processes Most of enzymes respond to changes of substrate concentration  an increase in the substrate conc. lead to increase in the rate of enzyme action  return the substrate conc. to normal level Some enzymes respond to allosteric effectors or covalent modification that affect the velocity of enzyme

Allosteric regulators (effectors) -Effectors that inhibit enzyme activity are termed negative effectors while those increase the enzyme activity are called positive effectors - Homotropic effectors: The substrate itself acts as effector, usually allosteric substrate are positive effector. The presence of substrate molecule at allosteric site will increase the catalytic activity of the substrate-binding site  sites cooperativity  Sigmoidal curve of V vs [S] not hyperbolic Heterotropic effectors The effectors are different from substrate Feed back inhibition A B C DE This enzyme has allosteric site that can bind to E The final product may have a feedback inhibition of on the enzyme that convert AB Feed back inhibition serves to coordinate the flow of substrate molecules through a series of reactions with the need of the cell.

Enzymatic Regulation

Covalent modification-Protein phosphorylation - Many enzymes may be regulated by covalent modification; addition or removal of phosphate group Phsphorylation occurs at the –OH of serine, threonine or tyrosine residues Phosphorylation process could be Activation or Deactivation process  depending on the enzyme itself Inactive or active

Induction and repression of enzyme synthesis Cells can regulate the amount of eth enzyme present, usually by altering the rate enzyme synthesis The induction or repression synthesis of protein leads to an alteration of the total number of enzyme population  increasing the number of active sites and Not affecting the enzyme activity.

The End

Learning Objectives Gibbs Free Energy Diagram – illustrates activation energy and two chemical steps Define Enzyme – protein, catalyst, specific, activates substrate Binding of Substrate to Active Center Transition State binds tighter than substrate or product Mechanism of Chymotrypsin – DHS triad and tetrahedral intermediate, swinging His

Learning Objectives Lineweaver-Burk plot Competitive, Uncompetitive and Noncompetitive Inhibition Possible models of inhibition Suicide and irreversible inhibition Natural inhibitors

Competitive inhibitors

Uncompetitive Inhibitors Inhibitor binds at an allosteric site, but only to the ES complex The slopes of 1/Vo vs. 1/[S] are unchanged but Vmax is lower, and so is the apparent [S] needed to reach 1/2 Vmax = Km. Vo

Enzyme inhibitors could be used as drugs Different types of enzyme inhibitors COMP UNCOMP NONCOMP