Biochemistry by Mary K. Campbell & Shawn O. Farrell

The Behavior of Proteins: Enzymes

Learning Objectives 1. What Makes Enzymes Such Effective Biological Catalysts? 2. What Is the Difference between the Kinetic and the Thermodynamic Aspects of Reactions? 3. How Do Substrates Bind to Enzymes? 4. What are the features of the active site ? 5. What Are Some Examples of Enzyme-Catalyzed reactions? 6. What Are Allosteric Enzymes ?  

Enzyme Catalysis Enzyme: a biological catalyst with the exception of some RNAs that catalyze their own splicing , all enzymes are globular proteins. enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction some enzymes are so specific that they catalyze the reaction of only one stereoisomer; others catalyze a family of similar reactions The rate of a reaction depends on its activation energy, DG°‡ an enzyme provides an alternative pathway with a lower activation energy (energy of activation)

Activation Energy Profile An enzyme alters the rate of a reaction, but not its free energy change or position of equilibrium

International Classifications of Enzymes Type of reaction catalyzed Class No. Transfer of electrons (hydride ions or H atoms) Oxidoreductases 1 Group transfer reactions Transferases 2 Hydrolysis reactions (transfer of functional groups to water) Hydrolases 3 Addition of groups to double bonds, or formation of double bonds by removal of groups Lyases 4 Transfer of groups within molecules to yield isomeric forms Isomerases 5 Formation of COC, COS, COO, and CON bonds by condensation reactions coupled to ATP cleavage. Ligases 6

Enzyme Catalysis Consider the reaction No catalyst Platinum surface Catalase 75.2 18.0 48.9 11.7 23.0 5.5 Activation energy (kJ/mol) (kcal/mol) Relative rate* 1 2.77 x 10 4 6.51 x 10 8 Reaction Conditions * Rates are given in arbitrary units relative to a value of 1 for the uncatalyzed reaction at 37°C

Units of energy A calorie (cal) is equivalent to the amount of heat required to raise the temperature of 1 gram of water from 14.5°C to 15.5°C. A kilocalorie (kcal) is equal to 1000 cal. A joule (J) is the amount of energy needed to apply a 1-newton force over a distance of 1 meter. A kilojoule (kJ) is equal to 1000 J. 1 kcal = 4.184 kJ

In an enzyme-catalyzed reaction substrate, S: a reactant that is converted into product by the enzyme active site: the small portion of the enzyme surface where the substrate (s) becomes bound. E + S ES enzyme-substrate complex

How the Enzyme Works? Enzymes are reusable!!!

Enzyme Catalysis Two models have been developed to describe formation of the enzyme-substrate complex lock-and-key model: substrate binds to the active site of the enzyme with a complementarily in shape. The active site is inflexible induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit It assumes flexibility of the enzyme The active site has a different 3D shape before and after substrate binding

Lock and Key Model

Induced Fit Model

The Active Sites of Enzymes Have Some Common Features 1. The active site is a three-dimensional cleft formed by groups that come from different parts of the amino acid sequence 2. The active site takes up a relatively small part of the total volume of an enzyme. 3. Active sites are clefts or crevices.

4. Substrates are bound to enzymes by multiple weak attractions electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions mediate reversible interactions of biomolecules. 5. The specificity of binding depends on the precisely defined arrangement of atoms in an active site.

Binding of a substrate to an enzyme at the active site. The enzyme chymotrypsin, with bound substrate in red

Chymotrypsin The Catalytic Triad. The catalytic triad, shown on the left, converts serine 195 into a potent nucleophile, as illustrated on the right.

Strategy and tactics. Chess and enzymes have in common the use of strategy

Enzymes commonly employ one or more of the following strategies to catalyze specific reactions Covalent catalysis: In covalent catalysis, the active site contains a reactive group, usually a powerful nucleophile that becomes temporarily covalently modified in the course of catalysis. The proteolytic enzyme chymotrypsin provides an excellent example of this mechanism

2. General acid-base catalysis: In general acid-base catalysis, a molecule other than water plays the role of a proton donor or acceptor. Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine

3. Metal ion catalysis. Metal ions can function catalytically in several ways. For instance, a metal ion may serve as an electrophilic catalyst, stabilizing a negative charge on a reaction intermediate. Alternatively, the metal ion may generate a nucleophile by increasing the acidity of a nearby molecule, such as water in the hydration of CO2 by carbonic anhydrase A nucleophile is a chemical species that donates an electron pair to an electrophile to form a chemical bond in relation to a reaction.

4. Catalysis by approximation: Many reactions include two distinct substrates. In such cases, the reaction rate may be considerably enhanced by bringing the two substrates together along a single binding surface on an enzyme.

Enzyme Catalysis: example Chymotrypsin catalyzes the selective hydrolysis of peptide bonds where the carboxyl is contributed by Phe and Tyr it also catalyzes hydrolysis of the ester bond of p-nitrophenyl esters

Kinetics of Chymotrypsin Catalysis Kinetics of Chymotrypsin Catalysis. Two stages are evident in the cleaving of N-acetyl-l-phenylalanine p-nitrophenyl ester by chymotrypsin: a rapid burst phase (pre-steady state) and a steady-state phase.

Note the hyperbolic shape of the curve

Proteases Facilitating a Difficult Reaction Protein turnover is an important process in living systems . Proteins that have served their purpose must be degraded so that their constituent amino acids can be recycled for the synthesis of new proteins. Proteins ingested in the diet must be broken down into small peptides and amino acids for absorption in the gut. proteolytic reactions are important in regulating the activity of certain enzymes and other proteins

Proteolytic enzymes as an example Proteolytic enzymes as an example. In vivo, these enzymes catalyze proteolysis, the hydrolysis of a peptide bond.

In the absence of a catalyst, the half-life for the hydrolysis of a typical peptide at neutral pH is estimated to be between 10 and 1000 years. Yet, peptide bonds must be hydrolyzed within milliseconds in some biochemical processes.

Most proteolytic enzymes also catalyze a different but related reaction in vitro namely, the hydrolysis of an ester bond.

Allosteric enzymes The activities of regulatory enzymes are modulated in a variety of ways. Function through reversible, noncovalent binding of regulatory compounds called allosteric modulators or allosteric effectors, which are generally small metabolites or cofactors. Other enzymes are regulated by reversible covalent modification. Both classes of regulatory enzymes tend to be multisubunit proteins, and in some cases the regulatory site(s) and the active site are on separate subunits.

END Chapter 6