Download Enzyme basic concepts, Enzyme Regulation IIII

Enzyme basic concepts, Enzyme Regulation I­III Carmen Sato­Bigbee, Ph.D. Objectives: 1) To understand the bases of enzyme catalysis and the mechanisms of enzyme regulation. 2) To understand the role of regulatory enzymes in controlling metabolic pathways and cellular responses. 3) To discuss the biological role of isoenzymes and their use in clinical diagnosis. Resources: Lehninger et al, Principles of Biochemistry 2005, Marks et al., Basic Medical Biochemistry, 2005 Enzyme Function Enzymes are proteins that catalyze specific biological reactions. “Catalyze” means that they increase the rate at which reactions reach equilibrium (they do not change the equilibrium constant of a reaction). For a reaction to take place, the molecules must interact with sufficient energy to produce a change resulting in the formation of different molecules. This involves the braking and making of chemical bonds with the formation of a high energy transition state that is reached using a minimum energy or energy of activation (Ea) that is required for a reaction to occur. Enzymes speed up reactions by lowering the Ea. Figure 1 ­ Original Advantages of biological reactions being catalyzed by enzymes: ­ Speed: At the temperature and pH present in cells and in the absence of enzymes, most chemical reactions would not occur at rates high enough to support life. Enzymes speed up reactions by a factor of ~10 6 to 10 14 .
­ Specificity: Enzymes are very specific for the type of reaction they catalyze and the substrates they utilize allowing only the “right” reactions to occur. ­ Control: Enzymes that catalyze crucial steps in biological processes are usually subjected to regulation by hormones, neurotransmitters and other factors. Thus, particular metabolic pathways and reactions will occur according to the metabolic requirements of the body/tissue at a particular time. Many diseases are actually caused by a faulty or absent enzyme or by a problem at the level of a regulatory mechanism. Classification and nomenclature of enzymes: Many enzymes were originally named by adding the suffix “ase” to the substrate’s name (for example, a protease catalyzes the addition of water to split a protein into smaller peptides). The International Union of Biochemistry (IUB) established a systematic classification of enzymes into six groups, according to the type of reaction catalyzed: Class 1: Oxidoreductases: catalyze oxidation and reduction reactions (dehydrogenases, reductases, oxidases, peroxidases, oxygenases, hydroxylases, catalases) Class 2: Transferases: transfer functional groups within the same molecule or from one molecule to another one (amino­, acyl­, methyl­, phosphoryl­ and glucosyltransferases; kinases; phosphomutases; transaldolase; transketolase) Class 3: Hydrolases: cleave a molecule into two molecules using water (glycosidases, esterases, peptidases, thiolases, amidases, phospholipases, ribonuclease, deoxyribonuclease) Class 4: Lyases: addition of groups to double bonds or formation of double bonds (decarboxylases, hydratases, aldolases, dehydratases) Class 5: Isomerases: catalyze intramolecular rearrangements resulting in formation of isomers (mutases, epimerases, cis­ and trans­isomerases) Class 6: Ligases: join two molecules creating a new bond at the expense of hydrolysis of a high energy bond such as in ATP or GTP (synthases). Each of these classes is divided into subclasses according to the chemical group or bond involved and into further subclasses according to other criteria. For example: EC 2.7.1.2 EC: Enzyme Commission; 2: transferase; 7: transfers a phosphate group; 1: an alcohol group accepts the phosphate; 2: denotes that the enzyme is ATP:D­glucose­6­ phosphotransferase. However, we will see later this enzyme with the common name of glucokinase. Enzyme Unit: amount of activity that catalyzes the transformation of 1 umol of substrate/minute. Specific activity: number of enzyme units/mg protein (umoles substrate/min/mg protein). Reaction velocity: amount of substrate modified/minute (umoles susbtrate/min).
Active site of enzymes The active or catalytic site of an enzyme is the region of the protein that binds the substrates and contains the amino acid residues (functional groups) that directly participate in the formation and braking of bonds. The active site may also bind to cofactors (metals, organic compounds) that also participate in the reaction. Functional groups within the active site of the enzyme stabilize the transition state complex and decrease the energy required for its formation. The active site has a “three­dimensional structure” formed by amino acids that are not necessary adjacent in the primary structure of the enzyme. Inhibitors or activators could modify the activity of an enzyme by altering the conformation of the protein. Enzyme specificity The specificity of enzymatic reactions depends on the precisely defined arrangement of atoms in the active site. The interaction between the enzyme and a substrate depends on the proper three­dimensional distribution of multiple electrostatic bonds, hydrogen bonds, and hydrophobic interactions. “lock­and­key” model: the active site of the free unbound enzyme is complementary in conformation to the unbound substrate. “Induced Fit” model: involves a process of dynamic recognition in which the conformation of the enzyme changes upon binding of the substrate in such a way that the active site and the substrate become complementary only after binding. Enzyme Kinetics and Regulation The kinetic properties of many enzymes follow the Michaelis­Menten equation: Vmax [S] V = _ Km + [S] In the Michaelis­Menten model the rate of the reaction is proportional to the concentration of the complex ES. k1 k2 E + S ø ES ÷ E + P The Michaelis­Menten equation relates the velocity (V) of an enzymatic reaction to the concentration of substrate S, Vmax and Km.
Vmax is the velocity of the reaction extrapolated to infinite concentration of S. What is Km? Km of an enzyme is the substrate concentration that gives a velocity equal to one­half of the maximal velocity. (V units are amount/time, [S] and Km units are concentration) By measuring the enzyme reaction at different substrate concentrations, the Michaelis­ Menten equation allows to determine experimentally the values of Km and Vmax for a particular enzyme. Figure 2 – Modified from Marks et al. The determination of the different parameters is facilitated if the Michaelis­ Menten equation is transformed into one that gives a straight­line plot. This equation can be obtained by taking the reciprocal of both sides of the Michaelis­ Menten equation: 1 1 K m 1 = ________ + _________ . _____ V Vmax Vmax [S] ____ The plot of 1/V versus 1/[S] is called Lineweaver­Burk plot and results in a straight line that intercepts the ordinate at 1/V = 1/Vmax . The slope is Km/Vmax. The intercept on the abscissa is –1/Km.
This type of plot is very useful for determining the kinetic of enzymes with various types of inhibitors. Figure 3­ Modified from Lehninger et al. Control of enzyme activity ­ Product concentration: The rate of enzymatic reactions “slows down” as products accumulate. The apparent decrease is due to increasing substrate formation by the reverse reaction as the product accumulates. In many cases the product works as an inhibitor. ­ Concentration of substrates and cofactors: In general enzymes evolved in a way that their Km values approximate the in vivo concentration of substrate. It is important to note that in vivo, a particular enzyme is not alone and is usually part of a pathway in which the activity of the preceding enzyme (the one that makes the substrate) or the one of the following enzyme (the one that uses the product) is critical in affecting the activity of the intermediate enzyme. ­ Genetic control: The “amount” of protein enzyme present in cells is in many cases under strict genetic control. Induction is the activation of enzyme synthesis. Repression is the inhibition of enzyme synthesis. Cells may respond to metabolic activity or extracellular signals by stimulating or inhibiting the transcription of a gene encoding a particular enzyme. ­ Covalent modification: Many enzymes can be either activated or inhibited by phosphorylation. The presence of phosphate groups (esterified to serine, threonine, tyrosine) can affect significantly the conformation of a protein. At the same time, the phosphorylating enzyme(s) (kinases) are subjected to stringent regulation by “second messengers” in signal transduction pathways. The phosphate groups can be removed by
phosphatases also subjected to regulation. We will see many examples that are crucial in regulating blood glucose levels. Many enzymes are synthesized as inactive precursors or zymogens that are then activated by cleavage at the proper time and place (digestive enzymes, blood coagulation cascade). Figure 4­ Taken from Marks et al. ­ Binding to proteins: Some enzymes can be regulated by binding to other proteins. For example Ca 2+ ­calmodulin is a protein that binds to kinases activating these enzymes (regulation of glycogen metabolism during muscle contraction). ­ Allosteric regulation: Allosteric enzymes are enzymes whose activity is modified by reversible non­covalent binding of specific modulators that bind to “allosteric sites” which are different from the catalytic sites. These modulators can be either activators or inhibitors. Allosteric enzymes do not follow Michaelis­Menten hyperbolic type kinetics, they exhibit sigmoideal kinetics. In allosteric enzymes the binding of modulators to one site can affect the properties of other sites in the same molecule. Most allosteric enzymes are oligomeric (several subunits). We will see some of these very important enzymes in carbohydrate metabolism
Figure 5­ Original Figure 6­ Modified from Lehninger et al. Enzyme inhibitors Inhibitors cause enzymatic reactions to occur more slowly. Enzymes subjected to the action of inhibitors may still obey the Michaelis­Menten kinetics but Km and Vmax values may vary with the inhibitor concentration.
Non­competitive inhibitors: These inhibitors may be reversible or irreversible and the effect is usually to decrease the activity of the enzyme by binding at a place other that the active site. The Km does not change but the Vmax is decreased. Examples: heavy­metal ions (Hg 2+ , Ag + , Pb 2+ ) that react with –SH groups in enzymes. Competitive inhibitors: These inhibitors resemble the substrate and compete with the substrate for the active site of the enzyme. This is equivalent to reduce the substrate concentration and therefore the enzyme shows an increased Km but the Vmax is not affected. Example: The enzyme Succinate dehydrogenase is inhibited by the competitor inhibitor malonate. Figure 7­ Taken from Lehninger et al.
Figure 8­ Modified from Lehninger et al. Feedback Inhibition: Generally involves allosteric enzymes and occurs when the end­product of a pathway inhibits its own synthesis. Usually the inhibitor inhibits the earlier steps in the pathway or reactions at a branching point. A very important way of saving cellular energy in biosynthetic pathways. Example: Negative regulation of glycolysis by ATP. Isoenzymes: Enzymes that catalyze the same reaction but differ in their amino acid sequence and properties. Usually these differences relate to different roles in different tissues, organs or developmental stages. Examples: glucokinase and hexokinase, both catalyze glucose phosphorylation but they have different Km (the biological importance of this problem will be discussed later in “Glycolysis”).
Lactate dehydrogenase (LDH), five different LDHs in different organs. Creatine kinase (CK), three forms in brain, muscle and heart. Both LDH and CK are used for the diagnosis of myocardial infarction. Isoenzymes 1) They catalyze the same reaction 2) They have different amino acid sequence and properties 3) Their characteristics may be related to their function in different tissues 4) They can be an important clinical diagnostic tool Figure 10­ Modified from Marks et al.
Isoforms of Lactate Dehydrogenase LDH 1 (H4): heart, kidney, red cells LDH 2 (H3 M): heart, kidney, red cells LDH 3 (H2 M2): brain, pancreas LDH 4 (H M3): lung, spleen LDH 5 (M4): skeletal muscle, liver Review questions 1) What are enzymes? 2) How do they work? 3) What is the biological advantage of using enzymes? 4) What is the active site of an enzyme? 5) How many classes of enzymes do you know? 6) What type of mechanisms of enzyme regulation do you know? 7) What is the Michaelis­Menten equation? 8) What kind of information can you obtain from a Lineaweaver­Burk plot? 9) What are allosteric enzymes? 10) How many types of enzyme inhibitors do you know, how can you distinguish them? 11) What is feedback inhibition? What is the biological relevance of this type of mechanism? 11) What are isoenzymes? Discuss their importance in clinical diagnosis.