Enzymes: the catalysts of life

All cellular reactions or processes are mediated by protein or RNA catalysts called enzymes. Protein enzymes are the topic of this chapter, however, RNAs are known to play more significant roles in biological processes.

G = -954 cal/mol

ivation energy and the metastable state

a chemical reaction can occur, the activation energy barrier must be overcome

ion energy EAis the minimum amount of energy required by the reactant molecules to collide

er and generate products

sition state, the reactants contain higher energy than their initially have. the state the reactants are

lecules with energy higher than EA can start reaction. stable state is a result of the activation energy barrier

e to inability to overcome the activation barrier. !table, with potential.

s overcome the activation energy barrier

activation vs. lowering EA with a catalyst.

2H2O2

2H2O + O2

ion "#e$+%& $',''' fold rate increase

se& ('',''',''' fold rate increase.

y (')*('(+ higher than non*catali,ed%

se contains iron atoms bound in chemical

Enzyme Stabilizes Transition State

ST

y change

EST

ES EP P

Energy decreases (under catalysis)

e basic properties of catalysts

Enzymes as biological catalysts

rease rate of reaction by lowering EA

m transient comple/ with the substrate molecules, but do not react with the substrate

nge the rate to achieve equilibrium, not the position of the equilibrium, i.e. 0eq is a constant.

mes are proteins !"As are catching

e site& "a% an actual groove or poc2et3 "b% in ion "4n, 5g, or #e% and other prosthetic electron acceptors3 "c% contains a few a.a. 67 of total a.a.%3

pecificity& inorganic catalysts vs. en,ymes

y to temperature& mostly $) o-3 !ome e at 8'o- "thermophilic achaebactria%.

Carboxypetidase

y to pH& pH affect charges of a.a. specially those at the active site. 9epsin h, pH23 1rypsin in intestine, pH8

Enzyme Classes

mes have been grouped into 6 general classes by The Enzyme Commission. These de 1) Oxidoreductases, 2) Transferases, 3) Hydrolases, 4) Lyases, 5) Isomerases ) Ligases. Since such classifications can aid recognition of enzymes, we will briefly ss each of these groups and provide examples.

xidoreductases: This is a very broad class of enzymes that catalyze the many

tion-reduction reactions found in biochemical pathways. Oxidoreductases catalyze ions in which at least one substrate gains electrons, becoming reduced, and another electrons, becoming oxidized.

mportant subset of oxidoctases are the dehydrogenases accept and donate electrons as ide ions (H:-) or hydrogen s often using cofactors such as +/NADH as an electron donor ceptor. An example is Lactate

LDH

ansferases: This class of enzymes catalyze

nsfer of a specific functional group between ules. Important subsets of transferases include ases that transfer phosphate groups, y from ATP to another molecule (such as inase and glucokinase, that phosphorylate se and protein kinases that phosphorylate n hydroyxl groups), b) Aminotransferases (see that transfer amino groups that are important in acid metabolism, c) Acyltransferases that er fatty acyl groups and d) syltransferases, which transfer carbohydrate es.
Aminotransferase reaction in which Pyridoxal phosphate (PLP) is used as a cofactor. (From Marks Basic Medical Biochemistry A clinical approach)

drolases: Hydrolysis reactions refer to the cleavage of bonds by the addition of a

ses: This class refers to those enzymes involved in cleaving bonds by means than hydrolysis or oxidation. Examples include aldolases (such as fructose sphate aldolase, which is involved in glycolysis) and thiolases (such as yl-CoA thiolase involved in the breakdown of fatty acids). Lyases also e enzymes involved in elimination of groups from two adjacent carbon atoms double bonds.

colysis, fructose 1,6sphate aldolase cleaves a n-carbon bond in fructose sphosphate. (From Marks Medical Biochemistry A al approach)

merases: At many stages of metabolism,

ngement of the atoms of a molecule is ed to create an isomer of the starting ound. Enzymes generally catalyzing the ngement of the bond structure are called rases, while those specifically catalyzing ovement of a phosphate from one group to er are known as mutases. For example, phosphate isomerase (right) catalyzes the onversion between dihydroxyacetone hate and D-glyceraldehyde 3-phosphate, is essential for continuing glycolysis ing splitting of six carbon sugars into two carbon sugars by fructose diphosphate se
(From Marks Basic Medical Biochemistry A clinical approach)

ases: Ligases are involved in synthesizing bonds between carbon atoms and n, nitrogen, oxygen or sulfur atoms in reactions that are coupled to the cleavage of

ate binding, activation, and reaction occur at the active site

ate binding transient, wea2 "$*(2 2cal:mol%, reversible

he loc2*and*2ey model "(8;+, #ischer%

he induced*fit model "(;68, 0oshland%

te activation

mation change in the en,yme& wea2en or distort bonds

a.a. electronegative group

rates

,yme may accept or donate protons and thus increase

mical reactivity of the substrates

,yme may also accept or donate electrons and

Enzymes $inetics

$inetics studies reaction rates and the effect of factors affecting the reaction ecially the concentrations of substrates, products and inhibitors.

refer to the initial reaction rates when the substrate concentrate has not tly increased and the accumulation of the product is low.

aelis%Menten $inetics indicates the hyperbola ip between initial reaction velocity "v% and the concentration <!=.

E!

2$

Ef + 9

v>

?ma/ <!=

0m + <!=

2+

Essential of Enzyme Kinetics

Steady State Theory

+
S

E +P

steady state, the production and consumption of

onstant ES Concentration at Steady State

S P

Concentration

ES

the ma/imum velocity and is proportional to concentration3 &hen substrate concentration is "<!= AA 0m%, the initial velocity is roughly ional to the substrate concentration

?ma/ <!=

0m + <!=

?ma/ <!=

0m

bstrate concentration is very high "<!= @@ 0m%

>

?ma/ <!=

0m + <!=

?ma/ <!=

<!=

> ?ma/

substrate concentration when the reaction at one*half of ?ma/.

>

?ma/ <!=

0m + <!=

>

?ma/ <!=

2<!=

> ?ma/ 2

Km: Affinity with Substrate

Vmax [S] vo = Km + [S] Vmax If vo = 2

hen using different substrate

S2

S1

Vmax Vmax [S] = 2 Km + [S]

4) BCbasics

S3

Km + [S] = 2 [S]

uble%reciprocal plot is a useful means of linearizing $inetic data

olic shape of 5ichaelis*5enten plot can not deduce the

?ma/ and hence 0m.

r way is to invert both side of the original 5ichaelis*

equation

v>

?ma/ <!=

0m + <!=

le e/periment& Eineweaver*Fur2 double reciprocal plot

A19

He/o2inase

glucose*C*phosphate + AD9

e concentration of one of the

nt, A19 to near saturate

graded concentration of

e in each tube from '.'6 to

a fi/ed amount of

inase and maintain the tube

m temperature "26o-%.

Enzyme Inhibition (Mechanism)

I

Competitive
E E
I
I

Non-competitive
S
I

Uncompetitive

Substrate

S S
Different site

E
S

I
I

Compete for Inhibitor active site

E + S ES E + P + I EI

E + S ES E + P + + I I EI + S EIS

E + S ES E + P + I EIS
[I] binds to [ES] complex

[I] binds to free [E] only,

[I] binds to free [E] or [ES]

s inhibitors act irreversibly or reversibly

ce: a control system activity regulation mechanisms of drug and poison functions& inhibiting en,yme functions ortant research tools such as using substrate analogues

ible inhibitors binds to en,yme covalently and irrevocably eliminate the catalytic activity of the yme. E.g.& heavy metal, nerve gas poison, and some insecticides.

le inhibitors

competitive inhibitors "binding to the same site as the substrate does% and competitive inhibitors "binding to another site on the surface of the en,yme%

Enzyme Inhibition (Plots)

I

I
Vmax

Competitive
I
Vmax

Non-competitive
I

Uncompetitive
Vmax Vmax

Vmax

vo

Km Km

[S], mM

Km = Km [S], mM

Km Km

[S], mM

Vmax unchanged Km increased 1/vo

Vmax decreased Km unchanged

Both Vmax & Km decreased 1/vo

1/vo

I
Two parallel lines

Intersect at Y axis

1/ Vmax

Intersect

1/ Vmax

1/ Vmax

Enzyme Inhibitors Are Extensively Used

ulfa drug (anti-inflammation)

Alzheimer's disease

Pseudo substrate competitive inhibitor

Protease inhibitor

Plaques in brain contains protein inhibitor

IV protease is critical to life cycle of HIV

subunit 1 subunit 2

IV protease (homodimer):

nhibitor is used to treat AIDS

As p

As p

Symmetry

En,yme regulation

e 2ey to en,yme regulation is conformational change.

both hemoglobin and he/o2inase, changes in ructure can have profound changes on the function activity of a protein molecule nformational changes can occur in response to teractions with other molecules, such as

!ubstrate,

substrate analogs, or pathway end*products Allosteric modulators -ovalently added groups Gegulatory subunits or accessory proteins 9roteolytic en,ymes

Regulation of Enzyme Activity

Proteolysis
proteolysis

tor

or

I
S

inhibitor

ack regulation

Phosophorylation

P
S

o
(+) P

regulator effector

phosphorylation

l transduction
A

A or

Enzymes regulation

level regulation

regulation directly depends on the interactions of substrates and products. E.g. 1he

vity of he/o2inase is inhibited by its product glucose*C*phosphate when over*produced.

ulation mechanisms& allosteric regulation and covalent modification

enzymes are regulated by molecules other than reactants and products

inhibition

A
E+

E(

E2

E$

regulation

en,ymes have two different forms, one with high affinity to the

strate and the other low "e.g. threonine deaminase%.

tory substance is called allosteric effector "e.g. isoleucine% and binds Allosteric activator

llosteric site.

Allosteric inhibitor

hy regulate en,yme activityI

egulation allows for efficient use of resources by the ll. JOnly ma2e it when you need itKJ etabolic pathways rarely stand alone, and usually tersect with numerous other pathways n en,yme pathways of several steps there are often ne "or more% 2ey en,ymes that are rate*limiting in ntrolling the flu/ through the pathway hey can respond to a variety of e/ogenous signals, hich in turn can increase or decrease their catalytic tivity, hus integrating e/pression of the pathway with other etabolic needs of the cell

5odel for cooperative binding

uential model&

ding of a ligand to a subunit affects the affinity the other subunits for the ligand. terms of binding constants& 0( @ 02 @ 0$ @ 0+. ere also is a Jconcerted model.J Foth together vide a description of the process.

osteric en,ymes diverge from 5*5 2inetics

gmoid rather than erbolic curve for a plot of s. <!= suggests allostery, ooperative interactions ng subunits ulating activators and:or bitors can cause maLor ts in these curves mechanism is implied ts may be in response to covalent binding or to alent attachment of a ifying group

vo

Noncooperative (Hyperbolic)

igmoidal curve
CTP

ATP

Positive effector (ATP) brings sigmoidal curve back to hyperbolic Negative effector

Cooperative (Sigmoidal)

vo
Consequently, Allosteric enzyme can sense the concentration of

xaggeration of igmoidal curve ields a drastic igzag line that hows the On/Off oint clearly

echanism and Example of Allosteric Effect

Models
Allosteric site

Kinetics
R
(+)
S
S

Cooperation

elax tive)

vo

R
Allosteric site

Homotropic (+) Concerted

[S]

(+)

vo
S

T
S

(+)

Heterotropic (+) Sequential

[S]

ense

Heterotropic

mple& Allosteric Activation:.nhibition

steric modulators may directly to an en,yme . threonine dehydra* %, or indirectly to a ulatoryO subunit ich in turn activates "or bits% the catalytic unit ome cases, allosteric ects are mediated by alent modification of the yme

/ample& #eedbac2 inhibition

c2 inhibition occurs when a late or ate product of a multi*step pathway its an early en,yme in the pathway ally at a rate*limiting step%. inhibition by isoleucine is steric "at a site on E( remote from active site%, and cific, in that nly isoleucine inhibits E( none of the ther intermediates do, and soleucine inhibits only E( not any of he other en,ymes "E2 E6%

enzymes e)hibit cooperative interactions bet'een

ric en,ymes are large, multisubunit proteins with an active site or

steric site on each subunits

tivity means that the whole protein undergoes confirmation change

either wor2ing sites are occupied.

erativity vs. negative cooperativity

an also be regulated by the addition of

chemical groups * covalent modification& addition of

hate groups, methyl groups, acetyl groups, or derivatives of

tides

tion,-ephosphorylation

egraded by phosphorylase by adding a phosphate group to the last

f a glycogen chain.

of phosphorylase is also regulated by phophorylation: ation. 9rotein 2inases add phosphate groups to other proteins or phosphorylation is cataly,ed by phosphoprotein phosphatases, e.g. sphatase

thase that adds glucose units to the glycogen chain is

ted in an opposite manner, i.e. phosphorylated form

9hosphorylation

e most common reversible covalent modification of oteins ds two negative charges, altering electrostatic interactions osphate groups also are effective at hydrogen bonding. orphorylation may alter substrate binding and:or catalytic tivity rge negative B for phosphorylation. About half of the *6' :mol is conserved in the phosphorylated protein "the other lf goes to ma2ing the reaction irreversible%. us phosphorylation can change conformational equilibria by rders of magnitude osphorylation is a highly effective means of regulation

Phosphorylation
Fischer, Kreb (1978)

r Thr Tyr (His)

OH

Kinase

phosphorylation
Conformational Change

Protein

dephosphorylastion

Phosphatase
Glycogen phosphorylase a

gen phosphorylase b

active

Active

Blycogen phosphorylase

rconversion between a and b s of the en,yme adds or ves phosphate from a serine ue to control its activity catalytic activity correlates differences in their ndary, tertiary, and ernary structures 2inase and phosphatase that y out these modifications ond in turn to the metabolic ands of the cellK part of a comple/ cascade way that mobili,es the energy ed in glycogen as it is needed.

Blycogen phosphorylase a form

Blycogen phosphorylase

phorylation at !er(+ in onse to epinephrine cle%:glucagon "liver% abili,es an M*terminal ent from an acidic to a c environment

terically activated by "indicating A19 levels ow%, noncovalently

ddition of glucose e/poses er(+ 9 to phosphatase,

Geversible covalent modifications

Geversible covalent modifications

teolysis also can play a regulatory role

4ymogens, or

proen,ymes, are inactive precursors of en,ymes. -leavage in the correct cellular compartment causes irreversible conformational changes that e/pose the active site.

Control Points of Gene Regulation

%ranscription DNA
"# process m N! $# mature m N! $# tail proteins

DNA

N! Processing N! %ransport N! &egradation

ribosome cap "#

N!

%ranslation

proteins

Activity

Proteolysis

Positive vs. Negative Regulation

-Galactosidase Activity in Response to Lactose

Organization of Lac Operon and LacI

promoter

Ribosome initiation

Regulation of Gene Expression

!"A molecules as enzymes: ribozymes

al/ 0120 sho'ed that the ron removal of the trahymena pre%r!"A is alyzed by itself

p formation tac$ by a hydro)yl group of a e guanosine that acts as a actor avage and splice and release intron ther autocatalytic to remove ther 01 nucleotides

et al/ sho'ed that the function ribonuclease + , a protein * A comple) for processing t!"A cursors lies in the !"A ponent, instead of the protein