Recitation 2014 Chapter 6 Lehninger

What are enzymes?

Enzymes are catalysts
Increase reaction rates without being used up
Most enzymes are globular proteins
However, some RNA (ribozymes and ribosomal RNA) also catalyze
reactions
We will celebrate my inspiration, the Biochemist Louis Pasteur.

Why biocatalysis over inorganic catalysts?

Greater reaction specificity: avoids side products
Milder reaction conditions: conducive to conditions in cells
Higher reaction rates: in a biologically useful timeframe
Capacity for regulation: control of biological pathways

Reaction Coordinate Diagram

Reaction coordinate diagram.

The free energy of the system is
plotted against the progress of
the reaction S P. A diagram of
this kind is a description of the
energy changes during the
reaction, and the horizontal axis
(reaction coordinate) reflects the
progressive chemical changes
(e.g., bond breakage or
formation) as S is converted to
P. The activation energies, G,
for the S P and P S
reactions are indicated. G is
the overall standard free-energy
change in the direction S P.

How to Lower

Enzymes organize reactive groups into close proximity and proper orientation

Uncatalyzed bimolecular reactions

two free reactants single restricted transition state
conversion is entropically unfavorable
Uncatalyzed unimolecular reactions
flexible reactant rigid transition state conversion is
entropically unfavorable for flexible reactants
Catalyzed reactions
Enzyme uses the binding energy of substrates to organize
the reactants to a fairly rigid ES complex
Entropy cost is paid during binding
Rigid reactant complex transition state conversion is
entropically OK

Enzymatic Catalysis
Enzymes do not affect equilibrium (G)
Slow reactions face significant activation
barriers (G) that must be surmounted
during the reaction
Enzymes increase reaction rates (k) by
decreasing G

kB T G

k
exp
h RT

How to Do Kinetic Measurements

Experiment:
1) Mix enzyme + substrate
2) Record rate of substrate disappearance/product formation as a
function of time (the velocity of reaction)
3) Plot initial velocity versus substrate concentration.
4) Change substrate concentration and repeat

Effect of Substrate Concentration

Effect of substrate concentration on the

initial velocity of an enzyme-catalyzed
reaction. The maximum velocity, Vmax, is
extrapolated from the plot because V0
approaches but never quite reaches Vmax.
The substrate concentration at which V0 is
half maximal is Km, the Michaelis constant.
The concentration of enzyme in an
experiment such as this is generally so low
that [S] >> [E] even when [S] is described
as low or relatively low. The units shown
are typical for enzyme-catalyzed reactions
and are given only to help illustrate the
meaning of V0 and [S]. (Note that the
curve describes part of a rectangular
hyperbola, with one asymptote at Vmax. If
the curve were continued below [S] = 0, it
would approach a vertical asymptote at [S]
= Km.)

Rate equation ;

Vmax [ S ]
v
Km S

Saturation Kinetics:
At high [S] velocity does not depend on [S]

Dependence of initial velocity on substrate

concentration. This graph shows the kinetic
parameters that define the limits of the curve at
high and low [S]. At low [S], Km >> [S] and the
[S] term in the denominator of the MichaelisMenten equation (Eqn 6-9) becomes
insignificant. The equation simplifies to V0 =
Vmax[S]/Km and V0 exhibits a linear dependence
on [S], as observed here. At high [S], where [S]
>> Km, the Km term in the denominator of the
Michaelis-Menten equation becomes
insignificant and the equation simplifies to V0 =
Vmax; this is consistent with the plateau
observed at high [S]. The Michaelis-Menten
equation is therefore consistent with the
observed dependence of V0 on [S], and the
shape of the curve is defined by the terms
Vmax/Km at low [S] and Vmax at high [S].

Lineweaver-Burk Plot:
Linearized, Double-Reciprocal

Enzyme Inhibition; although the term is enzyme inhibitor, often these are
chemotheraputic agents .
Inhibitors are compounds that decrease enzymes activit
Irreversible inhibitors (inactivators) react with the enzyme

One inhibitor molecule can permanently shut off one enzyme molecule
They are often powerful toxins but also may be used as drugs
Reversible inhibitors bind to and can dissociate from the enzyme

They are often structural analogs of substrates or products

They are often used as drugs to slow down a specific enzyme
Reversible inhibitor can bind:

to the free enzyme and prevent the binding of the substrate

to the enzyme-substrate complex and prevent the reaction
How do these inhibitors effect the protein structure, catalytic mechanism
and effect which kinetic values?
How do they appear to effect the graphic display of
Enzyme activity?

Chymotrypsin uses most of the enzymatic mechanisms, it is a

model for all serine protease
Structure of
chymotrypsin. (c)
The polypeptide
backbone as a ribbon
structure. Disulfide
bonds are yellow; the
three chains are
colored as in part (a).
(d) A close-up of the active site with a substrate (white and yellow) bound. The
hydroxyl of Ser195 attacks the carbonyl group of the substrate (the oxygens
are red); the developing negative charge on the oxygen is stabilized by the
oxyanion hole (amide nitrogens from Ser195 and Gly193, in blue), as
explained in Figure 622. The aromatic amino acid side chain of the
substrate (yellow) sits in the hydrophobic pocket. The amide nitrogen of the
peptide bond to be cleaved (protruding toward the viewer and projecting the
path of the rest of the substrate polypeptide chain) is shown in white.

Peptidoglycan and
Lysozyme
Hen egg white lysozyme and the
reaction it catalyzes. (b) Reaction
catalyzed by hen egg white
lysozyme. A segment of a
peptidoglycan polymer is shown,
with the lysozyme binding sites A
through F shaded. The glycosidic
CO bond between sugar residues
bound to sites D and E is cleaved,
as indicated by the red arrow. The
hydrolytic reaction is shown in the
Asp 52 acts as a nucleophile to attack the
inset, with the fate of the oxygen in
anomeric carbon in the first SN2 step
the H2O traced in red. Mur2Ac is NGlu 35 acts as a general acid and protonates
acetylmuramic acid; GlcNAc, Nthe leaving group in the transition state
acetylglucosamine. RO represents
a lactyl (lactic acid) group; NAc
Water hydrolyzes the covalent glycosyland AcN, an N-acetyl group (see
enzyme intermediate
key).
Glu 35 acts as a general base to deprotonate
water in the second SN2 step