The Kinetics of Enzyme Catalyzed Reactions

Dr. AKM Shafiqul IslamUniversity Malaysia Perlis

29.12.2009

Enzymes

• There are many chemical compounds in the living cell.

• How they are manufactured and combined at sufficient reaction rates under relatively mild temperature and pressure?

• How does the cell select exactly which reactants will be combined and which molecule will be docomposed?

• The answer is catalysis by enzyme.

Enzyme

Enzyme is protein or nucleic acid.• Chemical reactions

– breaking, forming and rearranging bonds.• Specificity

– Dictated by the enzyme active site.– Some active sites allow for multiple substrates.

• Cofactors– Amino acid side chains have a limited chemical repertoire.– Vitamin derivatives, metals (minerals) can bind as co-

substrates or remain attached through multiple catalytic cycles

Enzyme

Active Site

• Small relative to the total volume of the enzyme.

Crystal Structure of DNA Ligase

Catalysis

• Many reactions in biochemistry are spontaneous, meaning that they are thermodynamically favorable (DG<0). This does not mean, however, that they proceed rapidly.

• The oxidation of glucose to yield carbon dioxide and water is thermodynamically favorable (DG°’=-2870 kJ/mol).

• However, a jar of sugar, even in water, is incredibly stable, and has a half-life that is probably in the thousands of years in the absence of microbial contamination

Catalysts

• A catalyst is unaltered during the course of a reaction and functions in both the forward and reverse directions.

• In a chemical reaction, a catalyst increases the rate at which the reaction reaches equilibrium, though it does not change the equilibrium ratio.

• For a reaction to proceed from starting material to product, the chemical transformations of bond-making and bond-breaking require a minimal threshold amount of energy, termed activation energy.

• Generally, a catalyst serves to lower the activation energy of a particular reaction.

Enzymes• Proteins that assist in chemical reactions may be

Enzymes– Specific because of conformational shape

• Enzymes are catalysts– Catalyst: chemical that changes the rate of a reaction

without being consumed– Recycled (used multiple times)

• Enzymes reduce the activation energy of a reaction– Amount of energy that must be added to get a reaction

to proceed

Enzyme Nomenclature• active site - a region of an enzyme comprised of

different amino acids where catalysis occurs (determined by the tertiary and quaternary structure of each enzyme)

• substrate - the molecule being utilized and/or modified by a particular enzyme at its active site

• co-factor - organic or inorganic molecules that are required by some enzymes for activity. These include Mg2+, Fe2+, Zn2+ and larger molecules termed co-enzymes like nicotinamide adenine dinucleotide (NAD+), coenzyme A, and many vitamins.

Enzyme Nomenclature

• prosthetic group - a metal or other co-enzyme covalently bound to an enzyme

• holoenzyme - a complete, catalytically active enzyme including all co-factors

• apoenzyme - the protein portion of a holoenzyme minus the co-factors

• isozyme - (or iso-enzyme) an enzyme that performs the same or similar function of another enzyme. This generally arises due to similar but different genes encoding these enzymes and frequently is tissue-type specific or dependent on the growth or developmental status of an organism.

Enzymatic Reaction Principles

• Biochemically, enzymes are highly specific for their substrates and generally catalyze only one type of reaction at rates thousands and millions times higher than non-enzymatic reactions.

• Two main principles to remember about enzymes are a) they act as CATALYSTS (they are not consumed in a

reaction and are regenerated to their starting state) and b) they INCREASE THE RATE of a reaction towards

equilibrium (ratio of substrate to product), but they do not determine the overall equilibrium of a reaction.

Enzymatic Reaction Principles

• The energy maxima at which the reaction has the potential for going in either direction is termed the transition state.

• In enzyme catalyzed reactions, the same chemical principles of activation energy and the free energy changes (DGo) associated with catalysts can be applied.

• The overall negative DGo indicates a favorable reaction equilibrium for product formation.

• The net effect of the enzyme is to lower the activation energy required for product formation.

Reaction Rates• The rate of the reaction is determined by several factors

including the concentration of substrate, temperature and pH.

• For most standard physiological enzymatic reactions, pH and temperature are in a defined environment (pH 6.9-7.4, 37oC).

• This enzymatic rate relationship has been described mathematically by combining the equilibrium constant, the free energy change and first or second-order rate theory.

• The net result for enzymatic reactions is that the lower the activation energy, the faster the reaction rate, and vice versa.

Specificity

• Most synthetic catalyst are not specific i.e., they will catalyze similar reactions involving many different kinds of reactants. While enzymes are specific. They will catalyze only one reaction involving only certain substances.

• Enzyme specificity is thought to be a consequence of its three dimensional conformation which allows formation of the active site responsible for the catalytic ability of the enzyme.

INDUCED FIT

LOCK-AND-KEY

Binding Energy

• The graph of activation energy and free energy changes for an enzymatic reaction also indicates the role binding energy plays in the overall process.

• Due to the high specificity most enzymes have for a particular substrate, the binding of the substrate to the enzyme through weak, non-covalent interactions is energetically favorable and is termed binding energy.

• The same forces important in stabilizing protein conformation (hydrogen bonding and hydrophobic, ionic and van der Waals interactions) are also involved in the stable binding of a substrate to an enzyme.

Binding Energy and Transition State

• The cumulative binding energies from the non-covalent interactions are optimized in the transition state and is the major source of free energy used by enzymes to lower activation energies of reactions.

• A single weak interaction has been estimated to yield 4-30 kJ/mol energy, thus multiple interactions (which generally would occur during binding and catalysis) can yield up to 60-80 kJ/mol free energy - this accounts for the large decreases in activation energies and increases in rate of product formation observed in enzymatic-catalyzed reactions.

E + S ES EP E + PThis reaction proceeds spontaneously in the S to P direction.

Catalysis•Enzymes cannot change the equilibrium constant of any particular reaction, they can only speed the onset to equilibrium.

•The energy barrier between S and P, is called the activation energy, which is that required to reach the transition state. This energy reflects the formation of transient unstable charges, bond rearrangements, the alignment of reacting groups, and other transformations that are necessary for the reaction to proceed.

•The rate of a reaction correlates with the activation energy. The higher the activation energy (more unstable the transition state), the slower the reaction.

Potential-energy curves for the reaction of substrate, S, to products, P.

Comparison of activation energies in the uncatalyzed and catalyzed decompositions of ozone.