University:
Kentucky State University
Course:
CHE 315 | Biochemistry
Academic year:

2021
Views:

379

Pages:

35
Author:

Kundulu8mbj

Biochemistry 1 • Biochemistry of Amino Acids • Biochemistry of Proteins • Portrait of Allosteric Protein • Enzymes 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. • General points to be covered: 1. Enzymes are catalysts for biological reactions 2. They lower the activation energy 3. They increase the rate of reaction 4. Their activity is lost if denatured 5. They can be either: simple proteins or 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 1. Oxidoreductoases oxidation-reduction 2. Transferases transfer group of atoms 3. Hydrolases hydrolysis 4. Lyases add/remove atoms to/from a double bond 5. Isomerases rearrange atoms 6. 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 is flexible, not rigid •Enzyme and active site adjust shape to bind substrate •Increases range of substrate specificity •The shape changes also improves 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 cofactors 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 1. isolate the reaction substrate or 2. product from each other and 3. to provide a special environment for a reaction 4. to organize these reactions Turnover number and catalytic efficiency -Enzyme-catalyzed reactions are highly efficient, proceeding from 103 – 108 times faster than uncatalyzed 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 do enzymes work? The mechanism of action of enzymes can be explained by two different modes A. Energy changes during the enzyme-catalyzed reaction, enzyme provide an energetically favorable reaction pathway different from unanalyzed reaction B. 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 is 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) (T*-A) A → T* → B The peak of energy represents the Transition state in which the highenergy 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 of T* 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 ✓ Maximum activity reached when all of enzyme combines with substrate Maximum activity Reaction Rate ✓ Increasing substrate concentration→ increases the rate of reaction (enzyme concentration is constant) substrate concentration concentration Maximum activity Vmax Reaction Rate Most of enzymes show hyperbolic dependence of velocity on substrate Vmax/2 Km substrate concentration Factors Affecting Enzyme Action: Temperature Little activity at low temperature • Rate increases with temperature (the velocity increased with temp 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 Optimum temperature the velocity Reaction Rate • Low Temperature High 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 • Narrow range of activity, and the pH optimum varies for different enzymes Reaction Rate •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 Optimum pH 3 5 11 pH 7 9 Each enzyme has its optimum pH Michaelis-Menten Model Michalis and Menten proposed model that accounts for most features of enzymecatalyzed reactions. In this model the enzyme reversibly binds its substrate to form ES complex that subsequently breaks down to product E+S k1 ES k2 E+P 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 Vmax [S] Rate (vo) = 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 each 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 V max[S] vo = km + [S] [S] >> Km V max[S] vo = km + very small no V max [S] vo = km V (velocity of reaction) is roughly proportional to [S]➔ the rate of the reaction is first order reaction The V is constant and equal Vmax. The rate of reaction is independent on the [S]➔ the reaction rate is Zero order V max[S] vo = km + [S] [S] << Km V max[S] vo = very small no + [S] V max [S] vo = [S] vo = V max Order Rate Equation Comments zero rate = k rate is independent of substrate concentration first rate = k[S] rate is proportional to the first power of substrate concentration 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 at high [S]. 1 Vο vo =Vmax [S] / (Km + [S]) (Michaelis-Menten Equation) 1 [S] However, if 1/Vο is plotted vs 1/[S] a straight line is obtained→LineweaverBurk plot (or Double reciprocal plot)→ used to calculate Km and Vmax and determine the mechanism of action of enzyme inhibitors 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

Biochemistry 1 Enzymes

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