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 G

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 (ΔG)

• Slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction

• Enzymes increase reaction rates (k) by

decreasing ΔG‡

k kBT

h

expG

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.)

SK

SVv

m

][max

Rate equation ;

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 Michaelis-Menten 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 enzyme’s 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 6–22. 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

C—O 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

inset, with the fate of the oxygen in

the H2O traced in red. Mur2Ac is N-

acetylmuramic acid; GlcNAc, N-

acetylglucosamine. RO— represents

a lactyl (lactic acid) group; —NAc

and AcN—, an N-acetyl group (see

key).

Asp 52 acts as a nucleophile to attack the anomeric carbon in the first SN2 step

Glu 35 acts as a general acid and protonates the leaving group in the transition state

Water hydrolyzes the covalent glycosyl-enzyme intermediate

Glu 35 acts as a general base to deprotonate water in the second SN2 step