Midterm 1 is tomorrow, Thursday, SC/CHCP 131, 6:00pm-8:00pm
Review session this evening with Chris: SC/CHCP 131, 6:00pm-8:00pm
Questions
Any questions on the material from the previous lecture?
Section 6.4: Diffusion-Controlled Reactions
There is a theoretical upper limit to the rate of reactions in solution, based
upon the diffusion coefficients of reactant molecules, which determines how often they
collide with each other
Under physiological conditions, this limit is around 108 to 109 M-1s-1
A few kinds of enzymes approach this limit, and some, incredibly, even pass it
In order to accelerate a reaction to the diffusion limit, an enzyme must essentially
catalyze a reaction between substrates for every collision that occurs
Under physiological conditions, the velocity is determined by the second-order rate constant kcat/Km,
so enzymes that catalyze at the diffusion limit have rate constants near this value
Some Upper Limit Second-Order Rate Constants
We'll look briefly at two of the enzymes listed above, triose phosphate isomerase and superoxide dismutase
Note that superoxide dismutase appears to have a second order rate constant that exceeds the diffusion limit!
Triose Phosphate Isomerase
Triose phosphate isomerase (TPI) catalyzes the interconversion between DHAP and G3P, one of the reactions in glycolysis
and gluconeogenesis
In the forward direction, the result of the reaction is the removal of hydrogens from C1 and addition
of hydrogens to C2
One proposed mechanism for the enzyme involves acid-base catalysis and proton-shuttling to effect the transfer
Proposed TPI Mechanism (1)
Proposed TPI Mechanism (2)
Superoxide Dismutase
An even faster example of catalysis is seen with the enzyme superoxide dismutase, which helps
to remove the toxic free radical anion superoxide, ·O2-, a by-product of
oxidative metabolism
Superoxide dismutase catalyzes the first step in a two-step reaction, in which 4 superoxide
radicals are converted to 2 molecules of O2 and 2 molecules of hydrogen peroxide (H2O2):
The enzyme uses a bound copper atom to remove the single electron, where the electron is used to
reduce the copper atom in one step, and then is released and transferred to product in the second step:
E-Cu2+ + ·O2- → E-Cu+ + O2
E-Cu+ + ·O2- + 2H+ → E-Cu2+ + H2O2
Catalysis at Warp Speed
The kcat/Km for superoxide dismutase at 25°C has been determined to be 2 x 109M-1s-1
This rate is faster than the expected association rate of substrates for typical diffusion rates
How can the catalytic rate exceed the diffusion rate?
An Electric Field Enhances Substrate Docking
Determination of the 3D structure of the enzyme provided an explanation: the copper-atom binding site,
shown in green, is surrounded by positively-charged residues, shown in blue
This creates an electric field which attracts the negatively-charged substrate, allowing it to be drawn
in at faster-than-diffusion rates
Section 6.5: Binding Modes of Enzymatic Catalysis
Measurements have shown that the chemical effects of enzymatic catalysis only account for increases of around
10 to 100 over the uncatalyzed rate
The remainder of the rate increase is due to binding effects. There are two main types of binding
effects that have been identified in enzyme catalysis:
The proximity effect, which brings about a reduction of entropy
Stabilization of transition state (perhaps the most important contributor to rate acceleration)
In addition to these, we'll also consider some related issues, including weak binding of substrates to
enzyme, transition state analogs and catalytic antibodies
The Proximity Effect
The proximity effect describes how the enzyme reduces the entropy of the reaction by localizing and orienting
the substrates upon binding
This localization increases the effective concentration of the substrates, making them more likely to
react. Jencks and others developed a quantitative means of expressing this increase by measuring the rates
of unimolecular and bimolecular reactions. They developed a variable called the effective molarity:
This ratio produced large values for chemically-similar reactions of single molecule vs. bimolecular
substrates, showing how localization on a single molecule can increase the reaction rate
Experimental Demonstration of the Proximity Effect
Additional support for this rate increase was shown in experiments by Bruice and Pandit, involving the
2-step hydrolysis of p-bromophenyl acetate:
Weak Binding of Substrates
Although binding of substrate to enzyme is a major contributor to the rate increase, it's important
that the substrate not bind too tightly. Excessive ES stability actually prevents attainment of the
transition state, ES*
This excess stability is called a thermodynamic pit, as can be seen in a free energy reaction curve:
Consequently, the Km values of enzymes have evolved to reach an optimum between weak binding
and excessive binding stability of substrate
Transition State Stabilization
The final piece of the puzzle in enzyme catalysis is stabilization of transition state. This
refers to the ability of the enzyme to define the chemical and steric environment that are optimal for the
transition state of the reaction, not just simple complementarity to the substrate
This is a modification of the original lock and key model of catalysis, where both the substrate
(and in some cases the enzyme) undergo small changes in shape and chemical state upon binding
Confirmation of this effect is seen with the binding of transition state analogs, which are
molecules that resemble the transition state of an enzyme-catalyzed reaction
Shown above is 2-phosphoglycolate, an analog of the transition state in the reaction catalyzed by
triose phosphate isomerase. This analog binds the enzyme 100 times more strongly than either substrate
Catalytic Antibodies
A further demonstration of the importance of the transition state in enzyme catalysis has been shown
through the development of catalytic antibodies
Catalytic antibodies are molecules derived from immune cells exposed to an antigen that resembles the
transition state of a reaction
For example, a synthetic phosponamidate resembling the tetrahedral intermediate of an amide was used
to generate antibodies that demonstrated catalytic activity
The antibody was able to catalyze the hydrolysis of an amide at a rate only 1/25 that of the
chymotrypsin-catalyzed reaction
This result shows how the extraordinary functional capabilities of certain enzymes can be
acquired in short amounts of time, in a remarkable demonstration of molecular evolution
In conjunction with ongoing advances in understanding the mechanisms of enzyme catalysis, the de novo generation of such
abzymes points the way towards molecular engineering of new drugs and therapies
