A Key to Regulating Metabolism is Controlling the Rates of Chemical Reactions

Rxn requires: a hard collision in the right spot! RXN RATE = # of collisions x fraction with correct energy x fraction with correct orientation Rxn requires: a hard collision in the right spot!

How Do Enzymes Catalyze Chemical Reactions? (Essay topic on Test) Rate of Rxn is Determined by EA Key Idea – Lower Gibbs Free Energy of Activation, EA (minimum energy for reaction to occur)

Rate of Reaction is determined by Free Energy of Activation, ∆G ∆G = ∆H - T∆S Manipulating Entropy (∆S) and Enthalpy of Activation(∆H) Entropy – manipulate # of collisions with correct orientation by holding molecules in molecules next to each other with correct orientation. Enthalpy – i) Weaken (stress) bonds to be broken ii) Stabilize charge buildup in transition state THINK ABOUT HOW THE INDUCED FIT MODEL HELPS US UNDERSTAND THE ABOVE

How do Enzymes Lower Activation Energy? Create a microenvironment conducive to reaction: 1) Bind substrates in correct orientation for reaction 2) Weaken bonds to be broken (stretching and bending bonds in substrates )\ 3) Stabilize shape of transition state and charge buildup in transition state

Figure 6.12 Energy profile of an exergonic reaction

Concept of Correctly Oriented Collision: To React Purple must collide with Black

How likely is it for two macromolecules to collide at random in the exact spot, with the correct orientation for the reaction?

Impact on Rate of Holding Two Reactants in close proximity in Correct Orientation Rate depends on random collision in correct orientation Molecules held near one another in correct orientation: Observed Rate increases by a factor of 104 to 106 Link to orientation factor- Overall Reaction

Stabilizing Geometry and Charge Buildup in the transition state (TS) is a Key to catalysis Stabilize TS → Lower Ea Enzymes bind substrates loosely, but transition state tightly Induced Fit - Enzyme active site is complementary to substrate but typically upon binding substrate enzyme conformation changes slightly to favor transition state –

Link to Transition State Link to Transition State organic demo Link to stabilize transition state

Geometry of Transition State and Charge Buildup of Transition State

Enzyme Active Site Is Deeper than Substrate Instead, active site on enzyme also recognizes substrate, but actually complementally fits the transition state and stabilized it. If Substrate binds to enzyme perfectly complementally, no further reaction occurs. X 酵素 與抗體的最大不同點，在於兩者對目標的結合區構形不一樣。抗體只是很專一性遞與抗原結合了，再來就沒有進一步動作；酵素則不但與其基質結合，活化區口袋會誘導基質變成中間過渡狀態，然後很快轉成生成物。 Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252

X Stickase Substrate Transition state Product If enzyme just binds substrate then there will be no further reaction X Lehninger 課本上面 Stickase 的假想酵素，是一個極佳的例子，請多琢磨。 注意 Stickase 與基質結合區的外型，並非直接與基質成互補關係，而是與其『中間過渡』狀態有較佳的構形互補。另外，酵素與基質的結合區大多深陷入分子裡面，是酵素作用的一大優勢與特點。 Enzyme not only recognizes substrate, but also induces the formation of transition state Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252

Stabilizing Charges in Transition State Specific Induced to transition state Reaction in Water, Neutral pH C O = N H Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252 Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.167 +d 棒棒脢 的催化動作，可分成數個步驟，催化機制就是一步一步地探討這些步驟。活性區首先獲得基質，並以正確方向結合之，然後以吸引力誘導棒棒的扭曲，造成折斷後完成催化反應。此一模型正確顯現催化機制的構形扭曲，以及調整空間方向的貢獻，但是對吸引力的作用，則較看不出來。 事實上在酵素活性區中，有許多酸鹼性的催化協助機制，可以幫助酵素或基質，產生更強的反應基團，而達快速反應。 以本頁下半圖為例，酸或鹼可分別對兩種基團進行強化反應性的修飾 (中間 Fast 兩圖)； 但是只有在酵素的活性區中，利用預先安排好的基團，同時對攻擊與被攻擊的基團進行強化修飾，才會得到最大的催化反應速率。 -d H O Slow E5-2

Stabilizing Charges in Transition State Specific Induced to transition state Reaction in Water Neutral pH N H + Acid catalysis ACIDIC pH , add H+ to O C O = N H Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252 C O = N H C O = N H Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.167 +d 棒棒脢 的催化動作，可分成數個步驟，催化機制就是一步一步地探討這些步驟。活性區首先獲得基質，並以正確方向結合之，然後以吸引力誘導棒棒的扭曲，造成折斷後完成催化反應。此一模型正確顯現催化機制的構形扭曲，以及調整空間方向的貢獻，但是對吸引力的作用，則較看不出來。 事實上在酵素活性區中，有許多酸鹼性的催化協助機制，可以幫助酵素或基質，產生更強的反應基團，而達快速反應。 以本頁下半圖為例，酸或鹼可分別對兩種基團進行強化反應性的修飾 (中間 Fast 兩圖)； 但是只有在酵素的活性區中，利用預先安排好的基團，同時對攻擊與被攻擊的基團進行強化修飾，才會得到最大的催化反應速率。 H O -d H O H O BASIC pH, remove H+ from H2O C - O Base catalysis Slow Fast Fast E5-2

Stabilizing Charges in Transition State ENZYME, STABILIZES BOTH + and - Specific Induced to transition state Acid-base Catalysis N H + Acid catalysis N H + C O = N H Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252 C O = N H C O = N H C O = N H Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.167 +d Both 棒棒脢 的催化動作，可分成數個步驟，催化機制就是一步一步地探討這些步驟。活性區首先獲得基質，並以正確方向結合之，然後以吸引力誘導棒棒的扭曲，造成折斷後完成催化反應。此一模型正確顯現催化機制的構形扭曲，以及調整空間方向的貢獻，但是對吸引力的作用，則較看不出來。 事實上在酵素活性區中，有許多酸鹼性的催化協助機制，可以幫助酵素或基質，產生更強的反應基團，而達快速反應。 以本頁下半圖為例，酸或鹼可分別對兩種基團進行強化反應性的修飾 (中間 Fast 兩圖)； 但是只有在酵素的活性區中，利用預先安排好的基團，同時對攻擊與被攻擊的基團進行強化修飾，才會得到最大的催化反應速率。 H O H O -d H O H O C - O C - O Base catalysis Slow Fast Fast Very Fast E5-2

Active Site Avoids the Influence of Water + - 活性區 深埋在內部的一個重要原因是，催化反應必須避開水分子，以免反應受到水合的干擾，產生適當的鍵結與質子或電子轉移 (水分子實在太厲害了)。 Preventing the influence of water sustains the formation of stable ionic bonds Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115

Concerted Mechanism of Catalysis Carboxypeptidase A (248) Tyr O - H Active site pocket COO - (270) Glu 3 4 ACTIVE SITE Site for specificity O - H + H R N C COO - O - 5 2 協同式 的 carboxypeptidase 作用機制，請參考網頁上面的動畫，及講義上的文字說明。 (1) Zn2+ 離子乃重要輔助因子，可吸住基質胜肽鍵上的 carbonyl 基，增強其極性，使 (2) 碳帶正電。 (3) Glu 270 吸住水分子，放出 OH- 攻擊 C+ (2)，產生新的 C-OH 鍵。 (4) Tyr 248-OH 上的質子，與氮 lone pair 電子產生新鍵，原來的胜鍵斷裂。 (5) 附近的胺基酸與基質 C-端的 R 基團，有專一性的結合，以辨別基質的極性；同時 Arg 145 與基質 C-端的 -COOH 結合，確定基質蛋白質是以 C-端進入活性區。 Substrate peptide chain + Zn 1 +Arg (145) His (196) Glu (72) C-terminus Juang RH (2004) BCbasics Check for C-terminal His (69) E5-4

Enzyme Stabilizes Transition State Energy change ST Energy required (no catalysis) Energy decreases (under catalysis) EST S ES 有沒有 使用酵素催化的最大差別，在於過渡狀態的能量不同。由上圖可以看出在酵素催化下，到達過渡狀態的能量較低，也就是有酵素存在時，其過渡狀態比較容易形成。為什麼？最直接的原因是因為酵素可以穩定過渡狀態，因此反應物一下子就可跳到過渡狀態，然後很快以轉變成生成物。 那麼，為什麼酵素可以穩定過渡狀態？ P EP Reaction direction T = Transition state What’s the difference? Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166

Extra Negative Charge Was Neutralized -C-C-N-C-C-N-C-C-N- H H O - -C N- HO H O - -C N- HO H O -C N- H Chymotrypsin 如何穩定過渡狀態？ 可利用活性區附近蛋白質骨架上的 -N-H，因為此氫原子稍帶正電，可中和過渡狀態上的高密度負電荷 (氧)。 Chymotrypsin 的兩個反應階段，都產生類似的中間過渡狀態，也都可以利用相似的方式來穩定之。 E + S O -C-OH NH2- Adapted from Dressler & Potter (1991) Discovering Enzymes, p.179 E5-22

Practice IB Essay: Explain how enzymes catalyze chemical reactions Define enzyme Catalytic cycle (E + S, ES, EP, etc), Discussion of active site Key idea of catalysis (include labeled diagram of Free Energy vs Reaction Progress) Explain how enzyme accomplishes key idea