Chapter 11 Enzymatic catalysis

1. Chapter 11Enzymatic catalysis Role of enzymes • serve the same role as any other catalyst in chemistry • act with a higher specificity • acid and base catalyst possible due to proximal locations of amino acids • alter the structure location of quantity of the enzyme and you have regulated the formation of product - try that in organic chemistry ! • Don't lose sight of what enzymes do. Straight forward reactions…. reactants -> products.

2. Naming enzymes - • Enzyme commission for each enzyme based on the type of reactions. Kind of like IUPAC • Yeah right - the mother of invention – he/she who finds it names if. Trivial names rule

3. Enzyme reaction types: Enzymes are classified into six major classes based on the reaction they catalyze. 1) Oxidoreductases catalyze oxidation-reduction reactions. Most of these enzymes are known as dehydrogenases, but some are called oxidases, peroxidases, oxygenases or reductases. A- + B -> A + B- A is the reductant (e- donar) and B is the oxidant (e- acceptor) NAD+ (ox) +e- + H+ -> NADH (red) catalyzes the transfer of electrons from one molecule (the reductant, also called the hydrogen or electron donor) to another (the oxidant, also called the hydrogen or electron acceptor).

4. 2) Transferases catalyze group-transfer reactions (functional groups). Many require the presence of coenzymes. A portion of the substrate molecule usually binds covalently to these enzymes or their coenzymes. This group includes my personal favorite, the kinases A-X + B -> A + B-X A is the donar and B is the acceptor Donor s often a coenzyme ATP + R-OH -> ADP + R-PO4-2

5. 3) Hydrolases catalyze hydrolysis. They are a special class of transferases, with water serving as the acceptor of the group transferred A-B + H2O -> A-OH + B-H ie Glycoside hydrolases (also called glycosidases) catalyze the hydrolysis of the glycosidic linkage to generate two smaller sugars. They are extremely common enzymes with roles in nature including degradation of biomass such as cellulose and hemicellulose Together with glycosyltransferases, glycosidases form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.

6. Lyases catalyze nonhydrolytic and nonoxidative elimination reactions, or lysis, of a substrate, generating a double bond. In other words these reactions catalyze group elimination to form double bonds. In the reverse direction, lyases catalyze addition of one substrate to a double bond of a second substrate. A lyase that catalyzes addition reaction in cells is often termed a synthase. Usually only need one substrate in one direction and two for the reverse! ATP -> cAMP + PPi

7. 5) Isomerases catalyze the isomerase reactions. catalyses the structural rearrangement of isomers Because these reactions have only one substrate and one product, they are among the simplest enzymatic reactions. A -> B Names of these enzymes are “substrate isomersase” Ex. - enoyl CoA isomerase catalyzes conversion of cis-double bonds of fatty acids at position 3 to trans double bonds at position 2. It has a special importance in metabolism of unsaturated fatty acids or some sort of mutase a to b form of carbohydrate

8. 6) Ligases catalyze ligation or joining, of two substrates. These reactions require the input of chemical potential energy of a nucleoside triphosphate such as ATP. i.e. bond formation coupled to ATP hydrolysis. Ligases are usually referred to as synthetases. Ab + C -> A-C + b Sometimes involve a hydrolysis A and C are both large polymers DNA ligase

9. General Information on Enzymology Enzyme nomenclature • Active site • Substrate vs. reactant • Prosthetic groups, coenzyme and cofactors • Activity vs. reaction • Suffix -ase Another important reminder - not all activity is on or off. Many times the enzyme has a low or constitutive activity that can be increased many times. It is a common mistake to think of enzymes being on or off.

10. Enzyme specificity - One of the most powerful actions of enzymes. • Group complementation - the ability to recognize specific regions of the substrate to align reactants with catalytic site. • Based on non-covalent molecular interactions. • Lock and key vs. induced fit - both occur. Induced fit takes place when binding of one part of the substrate to the enzyme alters the conformation of the enzyme to make a true "fit" • Binding does not mean activity

11. Enzyme specificity An enzyme can bind and react stereo-specifically with chiral compounds • This can happen due to a three point attachment • Binding can then only occur in one way and therefore the products are not a mixture.

12. How do enzymes work? • By reducing the energy of activation • This happens because the transition state is stabilized by a number of mechanisms involving the enzyme/protein • Without enzymes reactions occur by collisions between reactants or addition of various organic catalysts • The energy barrier between the reactants and product, is the free energy of activation. • The free energy (G) of the reaction is what determines if a reaction is spontaneous.

13. How do enzymes work? • The rate is determined by That's why an enzyme can increase the speed / rate of a reaction. • The overall free energy of a reaction, G, is independent of the free energy of activation. So if the G is less than zero the reaction will proceed once a catalyst is added (the enzyme). • But the converse is also true - the reverse reaction is also able to proceed depending on the G. Some reactions have very little changes free energy and are freely reversible. Availability of enzyme (catalyst) and the relative concentrations of products and reactants will decide which direction these reactions will go.

14. Factors effecting catalysis Temperature – Unlike conventional chemical reactions, increasing temperature of a reaction can have mixed results.  Initial increases in temperature result in higher rates of reaction due to increasing the kinetic activity of reacting molecule – more forceful collisions and higher rates of diffusion between substrate and its enzyme  Increased temp exceeds energy barrier to maintain hydrogen and hydrophobic bonds of the enzyme. This results in denaturation of and loss of enzyme function

15. Factors effecting catalysis pH – There is typically an optimum pH of a reaction for both substrate and enzyme. The aa in the catalytic site as well as for structure is vital for the reaction to occur. Most enzymes are stable in the pH range of 5 – 9. Example - Enz- + SH+ -> EnzSH @ low pH Enz - -> EnzH @ high pH SH -> S- + H+

16. General catalytic mechanisms of enzyme reactions - also known as the exact means that enzymes lower the transition state and catalyze the reaction. • There are six general mechanisms by which enzymes act. These are the contributing factors of enzyme catalysis:

17. Acid-Base Catalysis Chemical groups can often be made more reactive by adding or removing a proton. Enzyme active sites contain side chain groups that act as proton donors or acceptors. These groups are referred to as general acids or general bases. The amino acid used in the reaction depends on the pH such that the side chain is in the appropriate charge state, and the local environment. Histadine is a good example of this. The side chain of histidine has a pKa of around 6. Therefore the side chain can undergo ionization at a physiological pH. The protonated form is a general acid and ionized form is the general base. The task of a catalyst is often to make a reactive group more reactive by increasing its intrinsic electrophilic or neutrophilic character. The easiest way to do this is to remove or add a proton. It is not practical to undergo this mechanism by free acids or bases and at physiological pH, the concentration of OH- and H+ is too low to induce the catalysis.

18. Therefore the correct placement of an amino acid that can act as a base or acid effectively increases the concentration at the correct location of the substrate. • See box 11-1 for pH effects on catalysis • RNase A is an example of this type of reaction

19. Covalent Catalysis In some enzymes a nucleophilic side chain group forms an unstable covalent bond with the substrate. The enzyme-substrate complex them forms product. The pathway can require that the intermediate is more susceptible to nucleophilic attack by water than the original substrate. • Three stages of covalent catalysis: • Formation of a bond between substrate and enzyme • Removal of electrons to make a reactive center • Elimination of the bond that was formed in step one

21. Metal Ion Catalysis Transition metals as well as divalent cations (Mg+2 and Ca+2) are useful for this type of catalysis. Metal ions act as a lewis acid and accept electrons. Therefore they are effective electrophiles. Another important reason for involving metals is the positive charge at any physiological pH. • Involved in redox reactions • Metals such as zinc activate water - to "acidify" or polarize the water so the OH group can act as a nucleophile. • Carbonic anhydrase is an example of this type of reaction

22. Zn2+ in carbonic anhydrase – Zn2+ is coordinated by 3 imidazole groups and a water molecule.(Arrow shows opening of active site)

23. Proximity and Strain (Orientation) Effects For a biochemical reaction to occur, the substrate most come into close proximity to catalytic functional groups (side chains of amino acids which are involved in catalytic reactions) with in the active site. In addition, the substrate must be precisely oriented to the catalytic groups. Once the substrate is correctly positioned, a change in the enzyme's conformation may result in a strained enzyme substrate complex. This strain helps to bring the enzyme substrate complex into the transition state. In general, the more tightly the active site can bind the substrate while it is in the transition state, the greater the rate of the reaction Proximity alone can only account for a five fold increase in activity Without the proper orientation, little reaction can occur - the Sn2 attack is a good example Lack of orientation is the key for an increased rate of reaction

24. Electrostatic Effects Recall that the strength of electrostatic interactions is related to the capacity of surrounding solvent molecules to reduce the attractive forces between chemical groups. Because water is largely excluded from the active site of most enzymes, the local dielectric constant is low. The charge distribution in the relatively anhydrous active site may influence the chemical reactivity of the substrate. In addition, weak electrostatic interactions such as those between permanent and induced dipoles in both the reactive site and the substrate are believed to contribute to catalysis. A more efficient binding of substrate lowers the free energy of the transition state, which accelerates the reaction. Thus enzymes act by stabilizing the distribution of electrical charge in the transition states. • Many inhibitors of enzymes mimic the transition state. The enzyme binds the inhibitor at a stable energy state - these inhibitors bind with a much higher affinity than the normal substrate.