Chapter 6 Enzymes

Significance of enzyme study:

1. Normal enzyme function is required for life maintenance

2. Medical treatment and diagnostic

3. Drug development

Aspartate aminotransferase (AST;SGOT) Alanine aminotransferase (ALT;SGPT)

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『雞尾酒療法』(Highly active antiretroviral therapy, HAART) ，於 1996 年由 何大一博士提出是指合併三種抗 HIV 病毒藥物 , 包括 蛋白酶抑制劑 (Protease Inhibitors) ＋非核苷類反轉錄酶抑制劑(Non-Nucleoside Reverse Transcriptase Inhibitors) ＋核苷類反轉錄酶抑制劑 (Nucleoside Reverse Transcriptase Inhibitors) 治療，以期降低病毒量、提高免疫力、改善存活率和減少抗藥種產生。雞尾酒療法藥物一個月大約要花費三萬元新台幣，一年大約花費 36萬。衛生署自 1997 年 4 月開始免費提供藥物，由指定醫院的感染科醫師負責開立處方，每位感染者及患者都可以在衛生署指定醫院獲得治療。

美國艾倫·戴蒙德艾滋病研究中心的主任

何大一博士

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Introduction to Enzymes

1897 Eduard Buchner --- yeast extracts can ferment sugar to alcohol

Frederick W. Kuhne --- the name “enzyme”

1926 James Sumner --- crystallization of urease John Northrop & Moses Kunitz --- crystallization of pepsin and trypsin J.B.S. Haldane --- treatise for “Enzymes” (weak-bonding interactions)

Most enzymes are proteins

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cofactorone or more inorganic ions

coenzymecomplex organic or metalloorganic molecule

prosthetic groupa cofactor or coenzyme tightly or covalently bound to enzyme

holoenzyme

apoenzyme (apoprotein)

Table 6-1

Table 6-2

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Enzymes are classified by the reactions they catalyze

Table 6-3

Phosphorylase b kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38)

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How enzymes work

Binding of a substrate to an enzymeat the active site

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Enzymes affect reaction rates, not equilibria

E + S ES EP E + P

Ground stateTransition state vs. reaction intermediate Activation energyRate-limiting stepC12H22O11 + 12 O2 12 CO2 + 11 H2O

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Reaction rates vs. Equilibria

K’eq = [P]/[S] G’o = -RT ln K’eq

V = k[S] = k [S1][S2] k = (k T/h)e-G /RT

Table 6-4 Table 6-5

1010, 100 億8

A few principles explain the catalytic power and specificity of enzymes

Binding energy (GB)--- the energy derived from enzyme-substrate interaction

1. Much of the catalytic power of enzymes is ultimately derived from the free energy released in forming multiple weak bonds and interactions between an enzyme and its substrate. This binding energy contributes to specificity as well as catalysis.

2. Weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to the substrate per se, but to the transition state through which substrates pass as they are converted into products during the course of an enzymatic reaction.

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Weak interactions between enzyme and substrate are optimized in the transition state

Dihydrofolate reductaseNADP+

tetrahydrofolate

Enzymes were structurally complementary to their substrates --- the “lock and key” model ---- Emil Fischer proposed in 1894

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In reality

A stickase

Induced fit

Lock and key

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Role of binding energy in catalysis

V = k [S1][S2] k = (k T/h)e-G /RT

V can be increased 10 fold when G decreased by 5.7 kJ/molFormation of a single weak interaction ~4 - 30 kJ/molBetween E and S, GB ~60 - 100 kJ/mol

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Table 6-5

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Binding energy vs. catalysis and specificity

Specificity --- the ability of enzymes to discriminate between a substrate and a competing molecule.

High specificity --- functional groups in the active site of enzyme arranged optimally to form a variety of weak interactions with a given substrate in the transition state

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Physical and thermodynamic factorsContributing to G , the barrier to reaction

Binding energy is used to overcome these barriers

1. The change in enthropy2. The solvation shell of H-bonded water3. The distortion of substrates4. The need for proper alignment of catalytic functional groups on the enzyme

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Rate enhancement by entropy reduction

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Specific catalytic groups contribute to catalysis(1) General acid-base catalysis

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Amino acids in general acid-base catalysis

102 to 105 order of rate enhancement 18

(2) Covalent catalysis

A B A + B

A B + X: A X + B A + X: + B

H2O

H2O

(3) Metal ion catalysis ionic interaction oxidation-reduction reactions

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Enzyme kinetics as an approach to understand mechanism

Enzyme kinetics --- determination of the rate of the reaction and how it changes in response to changes in experimental parameters

Fig. 6-11. Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction

V0 (initial velocity) when [S]>>[E], t is short

Vmax (maximum velocity) when [S]

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The relationship between substrate concentration and reaction rate can be expressed quantitatively

E + S ES E + Pk1

k-1

k2

V0 = k2[ES] Rate of ES formation = k1([Et]-[ES])[S] ---- (A) Rate of ES breakdown = k-1[ES] + k2[ES] ---- (B)Steady state assumption k1([Et]-[ES])[S] = k-1[ES] + k2[ES] ---- (A) = (B) k1[Et][S] - k1[ES][S] = (k-1 + k2)[ES] k1[Et][S] = (k1[S] + k-1 + k2)[ES] [ES] = k1[Et][S] / (k1[S] + k-1 + k2) divided by k1 [ES] = [Et][S] / {[S] + (k-1 + k2)/ k1} (k-1 + k2)/ k1 = is defined as Michaelis constant, Km

[ES] = [Et][S] / ([S] + Km)V0 = k2[ES] = k2[Et][S] / ([S] + Km) Vmax = k2[Et]V0 = Vmax [S] / ([S] + Km) Michaelis-Menten equation

* *

...

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V0 = Vmax [S] / ([S] + Km) Michaelis-Menten equation

When [S] = Km V0 = ½ Vmax

(When [S] is very small) (When [S] is very large)

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V0 = Vmax [S] / ([S] + Km)

1/V0 = Km /Vmax [S] + 1 /Vmax the double-reciprocal plot (Y = aX + b)

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Kinetic parameters are used to compare enzyme activities

Km = (k-1 + k2)/ k1 E + S ES E + Pk1

k-1

k2

if k2 << k-1 Km = k-1/ k1 = Kd Km relates to affinityif k2 >> k-1 Km = k2/ k1

if k2 ~ k-1

Table 6-6

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E + S ES E + Pk1

k-1

k2

Vmax = k2[Et]

kcat, the rate limiting of any enzyme-catalyzed reaction at saturation

kcat = Vmax / [Et] (turnover number)

Table 6-7

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V0 = Vmax [S] / ([S] + Km) M-M equation kcat = Vmax / [Et] Turnover number

V0 = kcat [Et] [S] / ([S] + Km) when [S] << Km ([S] is usually low in cells)V0 = kcat [Et] [S] / Km ( kcat / Km , specific constant)

kcat / Km has a upper limit (E and S diffuse together in aqueous solution)~108 to 109 M-1S-1 catalytic perfection

**Table 6-8

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Enzyme are subjected to inhibition (Reversible vs. irreversible inhibition)

1/V0 = Km /Vmax [S] + 1 /Vmax

(the double-reciprocal plot)

-1/Km

(a) Competitive inhibition(b) Uncompetitive inhibition(c) Mixed inhibition

When [I] ↑, Km? Vmax?28

1/V0 = Km /Vmax [S] + 1 /Vmax

1/Vmax

When [I] ↑, Km? Vmax?29

1/V0 = Km /Vmax [S] + 1 /Vmax

When [I] ↑, Km? Vmax?

1/Vmax

-1/Km

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Irreversible inhibition is an important tool in enzyme research and pharmacology

Chymotrypsin

Irreversible inhibitor

DIFP

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Suicide inactivator (mechanism-based inactivator)

These compounds are relatively unreactive until they bind to the active site of a specific enzyme.

Undergoes the first few chemical steps of the normal enzymatic reaction, but instead of being transformed into normal product, the inactivator is converted to a very reactive compound that combines irreversibly with the enzyme.

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Enzyme activity depends on pH

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Reaction mechanisms illustrate principles

chymotrypsin

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Amide nitrogens

AromaticSide chain

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Pre-steady state kinetic evidence for an acyl-enzyme intermediate

A40

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Colorless Yellow

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The pH dependence of chymotrypsin-catalyzed reactions

at low [S]

V0 = kcat [Et] [S] / ([S] + Km) when [S] << Km

([S] is usually low in cells)V0 = kcat [Et] [S] / Km

( kcat / Km , specific constant)

(pKa of R group of His57 = 6.0)

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(1)(2)

(3)

(4)

(5)

(6)

(7)

Reaction Mechanisms in the hydrolytic cleavage of a peptide bound by chymotrypsin

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(1)

(catalytic triad)

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(2)

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(3)

oxyanion hole

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(4)

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(5)

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(6)

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(7)

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(1)(2)

(3)

(4)

(5)

(6)

(7)

Reaction Mechanisms (the whole picture)

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Induced fit in hexokinase when binds to substrate

D-glucose

(H2O can go into the active site, but can not cause induced fit )48

Xylose is stereochemically similar to glucose, but can not be acted by hexokinase.

Xylose can cause induced fit of hexokinase, which “tricks” the enzyme to phosphorylate H2O

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The enolase reaction mechanism requires metal ions

Yeast enolase (MW ~93kDa), a dimer structure

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The two-step reaction catalyzed byEnolase in glycolysis

P (orange)

O (blue)

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(1) Effects of small structural changes in the substrate for chymotrypsin-catalyzed amide hydrolysis

Evidence for enzyme-transition state complementarity

If enzymes are complementary to reaction transition states, then some functional groups in both the substrate and the enzyme must interact preferentially in the transition state rather than in the ES complex.

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(2) Transition-state analogs/Catalytic antibodies

Ester hydrolysis Carbonate hydrolysis

Evidence for enzyme-transition state complementarity

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Regulatory enzymes

Allosteric enzymes vs. allorsteric modulators

Allosteric enzymes undergo conformational changes in response to modulator binding

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Two views of the regulatory enzyme aspartate transcarbamoylase(12 subunits)

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The regulatory step in many pathways is catalyzed by an allosteric enzyme

Feedback inhibition

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The kinetic properties of allosteric enzymes diverge from Michaelis-Menten behavior

+ Positive modulator- Negative modulator

S as a positive modulator

Vmax, Km

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Some regulatory enzymes undergo reversible covalent modification

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Phosphoryl groups affect the structure and catalytic activity of proteins

Glycogen phosphorylase

(Glucose)n + Pi (glucose)n-1 + glucose 1-phosphate

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AMP

P-Ser14

GlucosePLP

Regulation of glycogen phosphorylase60

Multiple phosphorylations allow exquisite regulatory control

OH

PO4

Proteinkinases

Proteinphosphatases

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Multiple regulatory phosphorylations

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Some types of regulation require proteolytic cleavage of an enzyme precursor --- zymogen

-S-S-

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The End

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Therapy of HIV Infection:Several distinct classes of drugs are now used to treat HIV infection:

1. Nucleoside-Analog Reverse Transcriptase Inhibitors (NRTI). These drugs inhibit viral RNA-dependent DNA polymerase (reverse transcriptase) and are incorporated into viral DNA (they are chain-terminating drugs). Zidovudine (AZT = ZDV, Retrovir) first approved in 1987 Didanosine (ddI, Videx) Zalcitabine (ddC, Hivid) Stavudine (d4T, Zerit) Lamivudine (3TC, Epivir)

2. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). In contrast to NRTIs, NNRTIs are not incorporated into viral DNA; they inhibit HIV replication directly by binding non-competitively to reverse transcriptase. Nevirapine (Viramune) Delavirdine (Rescriptor)

3. Protease Inhibitors. These drugs are specific for the HIV-1 protease and competitively inhibit the enzyme, preventing the maturation of virions capable of infecting other cells. Saquinavir (Invirase) first approved in 1995 Ritonavir (Norvir) Indinavir (Crixivan) Nelfinavir (Viracept)