Chapter 6 Enzymes Significance of enzyme study: 1. Normal enzyme function is required for life maintenance 2. Medical treatment and diagnostic 3. Drug.

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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) 1

『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 2

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 3

cofactor one or more inorganic ions coenzyme complex organic or metalloorganic molecule prosthetic group a cofactor or coenzyme tightly or covalently bound to enzyme holoenzyme apoenzyme (apoprotein) Table 6-1 Table 6-2 4

Enzymes are classified by the reactions they catalyze Table 6-3 Phosphorylase b kinase (ATP:phosphorylase phosphotransferase, EC ) 5

How enzymes work Binding of a substrate to an enzyme at the active site 6

Enzymes affect reaction rates, not equilibria E + S ES EP E + P Ground state Transition state vs. reaction intermediate Activation energy Rate-limiting step C 12 H 22 O O 2 12 CO H 2 O 7

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 , 100 億 8

A few principles explain the catalytic power and specificity of enzymes Binding energy (  G B )--- 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. 9

Weak interactions between enzyme and substrate are optimized in the transition state Dihydrofolate reductase NADP + tetrahydrofolate Enzymes were structurally complementary to their substrates --- the “lock and key” model ---- Emil Fischer proposed in

In reality A stickase Induced fit Lock and key 11

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/mol Formation of a single weak interaction ~ kJ/mol Between E and S,  G B ~ kJ/mol 12

Table

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 14

Physical and thermodynamic factors Contributing to  G, the barrier to reaction Binding energy is used to overcome these barriers 1. The change in enthropy 2. The solvation shell of H-bonded water 3. The distortion of substrates 4. The need for proper alignment of catalytic functional groups on the enzyme 15

Rate enhancement by entropy reduction 16

Specific catalytic groups contribute to catalysis (1) General acid-base catalysis 17

Amino acids in general acid-base catalysis 10 2 to 10 5 order of rate enhancement 18

(2) Covalent catalysis A B A + B A B + X: A X + B A + X: + B H2OH2O H2OH2O (3) Metal ion catalysis ionic interaction oxidation-reduction reactions 19

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 Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction V 0 (initial velocity) when [S]>>[E], t is short V max (maximum velocity) when [S]  20

21

The relationship between substrate concentration and reaction rate can be expressed quantitatively E + S ES E + P k1k1 k -1 k2k2 V 0 = k 2 [ES] Rate of ES formation = k 1 ([Et]-[ES])[S] ---- (A) Rate of ES breakdown = k -1 [ES] + k 2 [ES] ---- (B) Steady state assumption k 1 ([Et]-[ES])[S] = k -1 [ES] + k 2 [ES] ---- (A) = (B) k 1 [Et][S] - k 1 [ES][S] = (k -1 + k 2 )[ES] k 1 [Et][S] = (k 1 [S] + k -1 + k 2 )[ES] [ES] = k 1 [Et][S] / (k 1 [S] + k -1 + k 2 ) divided by k 1 [ES] = [Et][S] / {[S] + (k -1 + k 2 )/ k 1 } (k -1 + k 2 )/ k 1 = is defined as Michaelis constant, K m [ES] = [Et][S] / ([S] + K m ) V 0 = k 2 [ES] = k 2 [Et][S] / ([S] + K m ) V max = k 2 [Et] V 0 = V max [S] / ([S] + K m ) Michaelis-Menten equation * *... 22

V 0 = V max [S] / ([S] + K m ) Michaelis-Menten equation When [S] = K m V 0 = ½ V max (When [S] is very small) (When [S] is very large) 23

V 0 = V max [S] / ([S] + K m ) 1/V 0 = K m /V max [S] + 1 /V max the double-reciprocal plot (Y = aX + b) 24

Kinetic parameters are used to compare enzyme activities K m = (k -1 + k 2 )/ k 1 E + S ES E + P k1k1 k -1 k2k2 if k 2 << k -1 K m = k -1 / k 1 = K d K m relates to affinity if k 2 >> k -1 K m = k 2 / k 1 if k 2 ~ k -1 Table

E + S ES E + P k1k1 k -1 k2k2 V max = k 2 [Et] k cat, the rate limiting of any enzyme-catalyzed reaction at saturation k cat = V max / [Et] (turnover number) Table

V 0 = V max [S] / ([S] + K m ) M-M equation k cat = V max / [Et] Turnover number V 0 = k cat [Et] [S] / ([S] + K m ) when [S] << K m ([S] is usually low in cells) V 0 = k cat [Et] [S] / K m ( k cat / K m, specific constant) k cat / K m has a upper limit (E and S diffuse together in aqueous solution) ~10 8 to 10 9 M -1 S -1 catalytic perfection ** Table

Enzyme are subjected to inhibition (Reversible vs. irreversible inhibition) 1/V 0 = K m /V max [S] + 1 /V max (the double-reciprocal plot) -1/K m (a)Competitive inhibition (b)Uncompetitive inhibition (c)Mixed inhibition When [I] ↑, Km? Vmax? 28

1/V 0 = K m /V max [S] + 1 /V max 1/V max When [I] ↑, Km? Vmax? 29

1/V 0 = K m /V max [S] + 1 /V max When [I] ↑, Km? Vmax? 1/V max -1/K m 30

31

Irreversible inhibition is an important tool in enzyme research and pharmacology Chymotrypsin Irreversible inhibitor DIFP 32

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. 33

Enzyme activity depends on pH 34

Reaction mechanisms illustrate principles chymotrypsin 35

Amide nitrogens Aromatic Side chain 36

Pre-steady state kinetic evidence for an acyl-enzyme intermediate A 405 Colorless Yellow 37

The pH dependence of chymotrypsin-catalyzed reactions at low [S] V 0 = k cat [Et] [S] / ([S] + K m ) when [S] << K m ([S] is usually low in cells) V 0 = k cat [Et] [S] / K m ( k cat / K m, specific constant) (pKa of R group of His 57 = 6.0) 38

(1) (2) (3) (4) (5) (6) (7) Reaction Mechanisms in the hydrolytic cleavage of a peptide bound by chymotrypsin 39

(1) (catalytic triad) 40

(2) 41

(3) oxyanion hole 42

(4) 43

(5) 44

(6) 45

(7) 46

(1) (2) (3) (4) (5) (6) (7) Reaction Mechanisms (the whole picture) 47

Induced fit in hexokinase when binds to substrate D-glucose (H 2 O 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 H 2 O 49

The enolase reaction mechanism requires metal ions Yeast enolase (MW ~93kDa), a dimer structure 50

The two-step reaction catalyzed by Enolase in glycolysis P (orange) O (blue) 51

(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. 52

(2) Transition-state analogs/Catalytic antibodies Ester hydrolysis Carbonate hydrolysis Evidence for enzyme-transition state complementarity 53

Regulatory enzymes Allosteric enzymes vs. allorsteric modulators Allosteric enzymes undergo conformational changes in response to modulator binding 54

Two views of the regulatory enzyme aspartate transcarbamoylase (12 subunits) 55

The regulatory step in many pathways is catalyzed by an allosteric enzyme Feedback inhibition 56

The kinetic properties of allosteric enzymes diverge from Michaelis-Menten behavior + Positive modulator - Negative modulator S as a positive modulator Vmax, Km 57

Some regulatory enzymes undergo reversible covalent modification 58

Phosphoryl groups affect the structure and catalytic activity of proteins Glycogen phosphorylase (Glucose)n + Pi (glucose)n- 1 + glucose 1-phosphate 59

AMP P-Ser 14 Glucose PLP Regulation of glycogen phosphorylase 60

Multiple phosphorylations allow exquisite regulatory control OH PO 4 Protein kinases Protein phosphatases 61

Multiple regulatory phosphorylations 62

Some types of regulation require proteolytic cleavage of an enzyme precursor --- zymogen -S-S- 63

The End 64

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)