2 enzymes & enzyme kinetics

8/10/2019 2 Enzymes & Enzyme Kinetics

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The enzyme active site (features)

The catalytic site is relatively small

compared with the rest of the enzyme. Why

are many enzymes so big then? The catalytic site is a three-dimensional

entity

Substrates are bound to enzymes bymultiple weak, non-covalent interactions

(electrostatic bonds, hydrogen bonds, van

der Waals forces, hydrophobic interactions)


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Ribonuclease


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Catalytic sites form clefts or

crevices Substrate molecules bound within cleft

Water (unless involved in catalysis) is

normally excluded

Overall nonpolar character of cleft can

enhance binding of substrate

Cleft may also contain polar residues which

may take on catalytic properties within this

nonpolar microenvironment (exception to

the rule regarding hydrophobic core

present in many globular proteins)


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Active site involves amino acids far

apart in the primary sequence of a

protein (example: lysozyme)


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The specificity of binding depends

on the precisely defined arrangement

of atoms in an active site

Emil Fischer (over

100 years ago): came

up with the lock and

key hypothesis to

describe enzyme-substrate interactions


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Induced fit model: a

more refined model

that takes into account

the enzyme assumes a

complimentary shape

to that of its substrate

only after substrate

binds to the enzyme.

More dynamic

scenario compared tothe lock and key

hypothesis


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Michaelis-Menten model of

enzyme kinetics (Vmax & Km) Key element in their model is the existence

of the ES complex

Rate of catalysis (V) increases with

increasing [S], where V is defined as the

number of moles of product formed per

second


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When enzyme concentrations are constant,

V is linearly proportional to [S] WHEN [S]

IS SMALL.

At high [S] (when S is in vast excess of the

[enzyme]), V is nearly independent of [S]


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The Michaelis-Menten equation


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Km & Vmax

Km = the Michaelis constant

Defined as the [substrate] at which the

reaction rate is half of its maximal value

Used to define relative affinity of an

enzyme for its substrate

The higherthe Km value, the lowerthe

affinity and vice versa


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Vmax: describes the maximal rate of

product formation when [S] is high (i.e., invast excess of enzyme).

Under such conditions all of the existing

pool of enzyme active sites are full

From Vmax an enzymes turnover number

can be determined (expressed as the number

of substrate molecules converted intoproduct per unit time)


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Double-reciprocal (lineweaver-

Burk) plot Used to calculate Km

& Vmax

Also used tocharacterize

mechanisms of

enzyme inhibition by

specific compounds

Data expressed as 1/V

versus 1/[S]: gives a

straight line


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Calculating Km and Vmax


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Competitive vs.

noncompetitive

inhibitors


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Competitive inhibitors

Y intercept the same regardless of whether

inhibitor is present or absent, BUT the slope

differs between the two lines


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Competitive inhibitors

Do not alter Vmax

Increase Km

Competitive inhibition can be overcome by

increasing substrate concentration

Block substrate binding to the active site of

an enzyme


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Examples of competitive inhibitors

Alcohol (alcohol dehydrogenase)

UpCA (RNase)

DHFR inhibitors (DNA metabolic inhibitor

of tumors)

Sulfa drugs (anti-bacterial drugs)

Physiological examples: feedback

inhibition, pancreatic trypsin inhibitor


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Enzyme inhibition & automobile

antifreeze Ethylene glycol (EG) is a constituent of

antifreeze

EG not toxic but is converted to oxalic acidwhich form crystals in the kidneys leading

to extensive tissue damage and renal failure


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First step of conversion of EG to oxalic acid

is its oxidation to an aldehyde by alcohol

dehydrogenase

This reaction inhibited by ethanol which

competes with EG for binding to the alcohol

dehydrogenase


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An example of a

typical competitive

inhibitor:

UpCA has a very

similar structure

to the genuine

substrate, but is

chemically unableto undergo reaction.

Inhibition of RNase by

UpCA

U f E i hibit ti d


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Folate (folic acid)

Transformation of folate to tetrahydrofolate catalyzed by dihydrofolate reductase:

Competitive inhibitors of dihydrofolate reductase used in cancer treatment(resemble folate, bind ~1000x tighter):

eventually leads to synthesis of thymine nucleotides (DNA metabolism)

Use of Enzyme inhibitors as anti-cancer drugs:


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Sulfa Drugs

Resemble PABA in

structure

Blocks metabolicactivity of bacteria

E l f th Ph i l i l ( l t ) R l f E I hibit


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Examples of the Physiological (regulatory) Role of Enzyme Inhibitors

Feedback inhibition: The end-product of a biochemical pathway is similar to the

starting product and may (competitively) bind to and inhibit one of the enzymes

in the pathway:

Another example of regulatory competitive


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Another example of regulatory competitive

inhibition: Inhibition

by Pancreatic Trypsin Inhibitor


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Noncompetitive inhibitors

Plots converge on the X axis in the

presence or absence of inhibitor


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Noncompetitive inhibitors

Do not alter Km

Decrease Vmax

Noncompetitive inhibition cannot be

overcome by adding excess substrate

Bind to a site outside of catalytic site of

enzyme and act by decreasing the turnover

number of an enzyme


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In noncompetitive inhibition why

is Vmax decreased while Kmremains unchanged?


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The inhibitor lowers the

concentration of functional enzyme

The remaining uninhibited enzyme

behaves like a more dilute solution of that

enzyme (assumes [inhibitor] is limiting)

In other words, the substrate can still bind to

enzyme alone or enzyme complexed with

the inhibitor. But only free enzyme will

catalyze the reaction.

Since the pool of free enzyme is lower in

presence of inhibitor, Vmax will also be

lower


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Irreversible Enzyme Inhibitors

Inhibitor becomes covalently linked to the

enzyme

Attachment often occurs at the active site

Examples: 5-fluorouracil, DIPF (nerve gas),

penicillin


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Suicide Inhibitors

Irreversible enzyme inhibitors

Participate in the enzymatic reaction like the

substrate At some point in the reaction they get stuck

and become permanently linked to the enzyme.

Example: 5-Fluorouracil, a suicide inhibitor

which targets thymidylate synthase and is used

in cancer treatement.


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5-Fluorouracil

TS cannot catalyzereaction

A d dl li ti f i ibl


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A deadly application of irreversible enzyme

inhibition DIPF (Nerve Gas)

DIPF becomes permanentlylinked to the active-site serineof serine proteases

The toxic effect comes frominactivation ofacetylcholinesterase

The normal function of thisserine protease is to digest theneuromuscular transmitter

acetylcholine When acetylcholinesterase is

inactivated acetylcholinepersists. This leads to muscleparalysis and death.

Enzyme inhibitors as anti bacterial drugs


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Enzyme inhibitors as anti-bacterial drugs

Penicillin


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Most Drugs

andtoxins are

enzyme

inhibitors:

2 enzymes & enzyme kinetics

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