Table of Contents

Fundamentals of Biochemistry

Biochem 380 - Fall 2006

Lecture 016


Outline

Announcements


Questions


Mechanisms of Enzymes

Aspects of Enzyme Catalysis


Section 6.1: Terminology of Mechanistic Chemistry


Nucleophilic Substitutions



Nucleophilic Substitutions



Cleavage Reactions

R3-C-H → R3-C:- + H+
R3-C-H → R3-C+ + H-

  • In the second type of cleavage, which is less common, the paired electrons are split, producing unstable free radicals:
R1O-OR2 → R1O· + ·OR2

Oxidation-Reduction Reactions

  • Another set of mechanisms is seen in oxidation-reduction reactions. Oxidation refers to a loss of electrons. Reduction refers to a gain of electrons

  • Because electrons are conserved, a loss is paired with a gain in an overall reaction, with one substance losing electrons and another gaining them

  • Forms of oxidation include dehydrogenation, addition of oxygen and removal of electrons

  • Dehydrogenation is the most common form of oxidation, with dehydrogenase enzymes making up a large percentage of the oxido-reductases

  • A typical dehydrogenation reaction is the conversion of lactate to pyruvate, which involves the cleavage of a C-H bond and transfer of a hydride ion to NAD+:

Section 6.2: Transition States


  • As we have already seen in protein folding, the stable arrangements of molecules are associated with energy minimums (local 'wells' in the multidimensional energy landscape)

  • For a molecule (or pair of molecules) to move from one stable arrangement to another, it must cross intermediate, higher-energy barriers in the energy landscape

  • Like protein folding, chemical reactions represent transitions from one energy state to another. The peaks of the barriers between substrate and product energies are termed the transition states of a reaction

A Macroscopic Analogy


  • One way to think about energy barriers between molecular states is to compare them to transitions in macroscopic objects

  • One of my favorite macroscopic objects is a canning jar that holds my coffee

  • The jar has a spring clamp mechanism that holds the lid tightly down over the top of the jar, in a stable 'closed' state

  • When the lever of the spring clamp is pushed out, the mechanism briefly enters a 'strained', high-energy transition state before acquiring a loose 'open' state

  • In a similar way, the bonds and electronic interactions between atoms in a molecule define 'strained' transition states between stable molecular configurations

Enzymes Lower the Activation Energy


  • For spontaneous reactions, the energy state of the products will be lower than that of the reactants (negative ΔG). For most reactions, the intermediate transition state will be higher than the energy level of the reactants (also called the 'ground' state)

  • The difference in energy between the ground state and the transition state is called the activation energy

  • In a number of ways, enzymes are able to accelerate the rate of a reaction by lowering the activation energy (the barrier to the progress of a reaction)

  • In the absence of enzyme, the reactants will only interact through random collisions. Out of the total set of collisions in a given time, only a small fraction will have the necessary velocities and orientations sufficient to 'cross' the transition state barrier

Section 6.3: Chemical Modes of Enzyme Catalysis

  • The chemical modes of catalysis refers to the chemical effects that an enzyme contributes to the reaction (in contrast to binding and conformational effects)

  • The two main types of chemical effects in enzymes are acid-base catalysis and covalent catalysis

  • Both of these effect are brought about through the use of polar and ionizable side chains in the enzyme. These can be thought of as the 'hot-spots' in the otherwise unreactive hydrophobic pocket that composes the active site of most enzymes

  • The following side chains act to provide the majority of reactive groups in enzyme catalysis:

Acid-Base Catalysis

  • Acid-base catalysis uses the transfer of a proton to accelerate bond-breaking and bond-formation in reactions. This kind of catalysis is common in both organic chemistry and in enyzme-catalyzed reactions

  • In enzymes, the ionizable groups of amino-acid side chains act as the general acids and bases that can donate and receive protons

  • In the reaction above, a general base, symbolized by B:, accepts a proton to cleave the C-H bond

  • A general acid, symbolized by BH+, can also catalyze cleavage by donating a proton to an acceptor, such as the oxygen of the hydroxyl group shown above

Covalent Catalysis

  • Covalent catalysis usually consists of a multistep reaction in which covalent bonds are formed between the enzyme and substrate to produce reactive intermediates, followed by bond-breaking to release the final products

  • For example, the transfer of a group X from molecule A to molecule B takes place in a two-step reaction:
A-X + E → A + E-X

E-X + B → E + B-X

  • This two-step sequence is also an important means for coupling the energy of one reaction to another. For example, covalent catalysis of sucrose (a disaccharide of glucose and fructose) occurs through a glucosyl-enzyme intermediate:

Sucrose + Enzyme → Glucosyl-Enzyme + Fructose

Glucosyl-Enzyme + Pi → Enzyme + Glucose-1-phosphate

pH Effects

  • The activity of ionizable residues in the active site of an enzyme can be identified through changes in pH

  • This can be done through the creation of a pH profile, in which the activity of the enzyme is assayed over a range of pH values:

  • Here, the activity of papain, a protease enzyme derived from the papaya fruit, is shown with a bell curve that results from the ionization of two key residues, a cysteine and a histidine

  • The curve shows the active range of the enzyme coinciding with the cysteine deprotonated and the histidine protonated

Active Form of Papain

Questions


  • Questions about the material covered today?

Next Lecture: Sections 6.4 - 6.5


  • Read Sections 6.4 - 6.5