定義
酶是用于生化反應的非常有效的催化劑。它們通過提供較低活化能的替代反應途徑來加快反應速度。酶作用于底物并產生產物。一些物質降低或什至停止酶的催化活性被稱為抑制劑。
發(fā)現
1965年，Umezawa H分析了微生物產生的酶抑制劑，并分離出了抑制亮肽素和抗痛藥的胰蛋白酶和木瓜蛋白酶，乳糜蛋白酶抑制的胰凝乳蛋白酶，胃蛋白酶抑制素抑制胃蛋白酶，泛磷酰胺抑制唾液酸酶，烏藤酮抑制酪氨酸羥化酶，多巴汀抑制多巴胺3-羥硫基嘧啶和多巴胺3-羥色胺酶酪氨酸羥化酶和多巴胺J3-羥化酶。最近，一種替代方法已應用于預測新的抑制劑：合理的藥物設計使用酶活性位點的三維結構來預測哪些分子可能是抑制劑1。已經開發(fā)了用于識別酶抑制劑的基于計算機的方法，例如分子力學和分子對接。
結構特征
已經確定了許多抑制劑的晶體結構。已經確定了三種與凝血酶復合的高效且選擇性的低分子量剛性肽醛醛抑制劑的晶體結構。這三種抑制劑全部在P3位置具有一個新的內酰胺部分，而對胰蛋白酶選擇性最高的兩種抑制劑在P1位置具有一個與S1特異性位點結合的胍基哌啶基。凝血酶的抑制動力學從慢到快變化，而對于胰蛋白酶，抑制的動力學在所有情況下都快。根據兩步機理2中穩(wěn)定過渡態(tài)絡合物的緩慢形成來檢驗動力學。
埃米爾•菲舍爾（Emil Fischer）在1894年提出，酶和底物都具有特定的互補幾何形狀，彼此恰好契合。這稱為“鎖和鑰匙”模型3。丹尼爾·科什蘭（Daniel Koshland）提出了誘導擬合模型，其中底物和酶是相當靈活的結構，當底物與酶4相互作用時，活性位點通過與底物的相互作用不斷重塑。
在眾多生物活性肽的成熟過程中，需要由其谷氨酰胺（或谷氨酰胺）前體形成N末端焦谷氨酸（pGlu）。游離形式并與底物和三種咪唑衍生抑制劑結合的人QC的結構揭示了類似于兩個鋅外肽酶的α/β支架，但有多個插入和缺失，特別是在活性位點區(qū)域。幾種活性位點突變酶的結構分析為針對QC相關疾病5的抑制劑的合理設計提供了結構基礎。
作用方式
酶是催化化學反應的蛋白質。酶與底物相互作用并將其轉化為產物。抑制劑的結合可以阻止底物進入酶的活性位點和/或阻止酶催化其反應。抑制劑的種類繁多，包括：非特異性，不可逆，可逆-競爭性和非競爭性?？赡嬉种苿?nbsp;以非共價相互作用（例如疏水相互作用，氫鍵和離子鍵）與酶結合。非特異性抑制方法包括最終使酶的蛋白質部分變性并因此不可逆的任何物理或化學變化。特定抑制劑 對單一酶發(fā)揮作用。大多數毒藥通過特異性抑制酶發(fā)揮作用。競爭性抑制劑是任何與底物的化學結構和分子幾何結構非常相似的化合物。抑制劑可以在活性位點與酶相互作用，但是沒有反應發(fā)生。非競爭性抑制劑是與酶相互作用但通常不在活性位點相互作用的物質。非競爭性抑制劑的凈作用是改變酶的形狀，從而改變活性位點，從而使底物不再能與酶相互作用而產生反應。非競爭性抑制劑通常是可逆的。不可逆抑制劑與酶形成牢固的共價鍵。這些抑制劑可以在活性位點附近或附近起作用。
功能
工業(yè)應用中， 酶在商業(yè)上被廣泛使用，例如在洗滌劑，食品和釀造工業(yè)中。蛋白酶用于“生物”洗衣粉中，以加速蛋白質在諸如血液和雞蛋等污漬中的分解。商業(yè)上使用酶的問題包括：它們是水溶性的，這使得它們難以回收，并且一些產物可以抑制酶的活性（反饋抑制）。
藥物分子，許多藥物分子都是酶抑制劑，藥用酶抑制劑通常以其特異性和效力為特征。高度的特異性和效力表明該藥物具有較少的副作用和較低的毒性。酶抑制劑在自然界中發(fā)現，并且也作為藥理學和生物化學的一部分進行設計和生產6。
天然毒物 通常是酶抑制劑，已進化為保護植物或動物免受天敵的侵害。這些天然毒素包括一些已知最劇毒的化合物。
神經氣體（ 例如二異丙基氟磷酸酯（DFP））通過與絲氨酸的羥基反應生成酯，從而抑制了乙酰膽堿酯酶的活性位點。
參考
1、Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.
2、Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.
3、Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.
4、Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.
5、Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.
6、Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

Definition
Enzymes are very efficient catalysts for biochemical reactions. They speed up reactions by providing an alternative reaction pathway of lower activation energy. Enzyme acts on substrate and gives rise to a product. Some substances reduce or even stop the catalytic activities of enzymes are called inhibitors.

Discovery
In 1965, Umezawa H analysed enzyme inhibitors produced by microorganisms and isolated leupeptin and antipain inhibiting trypsin and papain, chymostatin inhibiting chymotrypsin, pepstatin inhibiting pepsin, panosialin inhibiting sialidases, oudenone inhibiting tyrosine hydroxylase, dopastin inhibiting dopamine 3-hydroxylase, aquayamycin and chrothiomycin inhibiting tyrosine hydroxylase and dopamine J3-hydroxylase . Recently, an alternative approach has been applied to predict new inhibitors: rational drug design uses the three-dimensional structure of an enzyme's active site to predict which molecules might be inhibitors 1. Computer-based methods for identifying inhibitor for an enzyme have been developed, such as molecular mechanics and molecular docking.

Structural Characteristics
The crystal structures of many inhibitors have been determined. The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined. All the three inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism 2.

Emil Fischer in 1894 suggested that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.This is known as "the lock and key" model 3. Daniel Koshland suggested induced fit model where substrate and enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme 4.

N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors reveals an alpha/beta scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The structural analyses of several active-site-mutant enzymes provide a structural basis for the rational design of inhibitors against QC-associated disorders 5.

Mode of Action
Enzymes are proteins that catalyze chemical reactions. Enzymes interact with substrate and convert them into products. Inhibitor binding can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalyzing its reaction. There are a variety of types of inhibitors including: nonspecific, irreversible, reversible - competitive and noncompetitive. Reversible inhibitors bind to enzymes with non-covalent interactions like hydrophobic interactions, hydrogen bonds, and ionic bonds. Non-specific methods of inhibition include any physical or chemical changes which ultimately denature the protein portion of the enzyme and are therefore irreversible. Specific Inhibitors exert their effects upon a single enzyme. Most poisons work by specific inhibition of enzymes. A competitive inhibitor is any compound which closely resembles the chemical structure and molecular geometry of the substrate. The inhibitor may interact with the enzyme at the active site, but no reaction takes place. A noncompetitive inhibitor is a substance that interacts with the enzyme, but usually not at the active site.  The net effect of a non competitive inhibitor is to change the shape of the enzyme and thus the active site, so that the substrate can no longer interact with the enzyme to give a reaction. Non competitive inhibitors are usually reversible. Irreversible Inhibitors form strong covalent bonds with an enzyme.  These inhibitors may act at, near, or remote from the active site .

Functions
Industrial application, enzymes are widely used commercially, for example in the detergent, food and brewing industries. Protease enzymes are used in 'biological' washing powders to speed up the breakdown of proteins in stains like blood and egg. Problems using enzymes commercially include: they are water soluble which makes them hard to recover and some products can inhibit the enzyme activity (feedback inhibition) .

Drug molecules, many drug molecules are enzyme inhibitors and a medicinal enzyme inhibitor is usually characterized by its specificity and its potency. A high specificity and potency suggests that a drug will have fewer side effects and less toxic. Enzyme inhibitors are found in nature and are also designed and produced as part of pharmacology and biochemistry 6.

Natural poisons are often enzyme inhibitors that have evolved to defend a plant or animal against predators. These natural toxins include some of the most poisonous compounds known.

Nerve gases such as diisopropylfluorophosphate (DFP) inhibit the active site of acetylcholine esterase by reacting with the hydroxyl group of serine to make an ester.

References

Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.

Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.

Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.

Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.

Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.

Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

Definition

Caspases are a family of aspartate specific cysteine proteases that play an important role in apoptosis, necrosis and inflammation1.

Discovery

Caspases were first identified in the nematode C. elegans. It was found that the gene ced-3 was required for cell death during C.elegans development2. In 1993, the protein encoded by the ced-3 gene was identified as a cysteine protease and it was found that it had similar properties to the mammalian interleukin-1-beta converting enzyme (ICE) (now known as caspase 1) which at the time was the only known caspase3. Other mammalian caspases were subsequently identified.                                                                          

Classification

There are three types of apoptotic caspases: initiator, effector and inflammatory caspases. Initiator caspases (e.g. CASP2, CASP8, CASP9 and CASP10) cleave inactive pro-forms of effector caspases, thereby activating them4. Effector caspases (e.g. CASP3, CASP6 and CASP7) in turn cleave other protein substrates within the cell, to trigger the apoptotic process4. Inflammatory caspases are involved in immune response (e.g. CASP1, CASP4, CASP5, CASP11, CASP12 and CASP13). Caspase inhibitors regulate the initiation of this cascade4.

Structural Characteristics

Caspases are synthesized as inactive zymogens or procaspases.  Activation of caspases occurs by cleavage of the prodomain in the procaspases5.  The caspase catalytic domain is composed of a twisted, mostly parallel ß-sheet sandwiched between two layers of a-helices. Also they contain an active cysteine residue in their catalytic domain5.  In addition to the catalytic domain, both inflammatory and initiator caspases carry at their N-termini, one or two copies of CARD or DED modules, which are critical for their activation in vivo. These modules are mainly composed of six antiparallel a-helices, with helices a1–a5 building an a-helical Greek key5. The general structure of a caspase inhibitor is [tetrapeptide]-CO-CH2-X, that binds to the Cys285 in the active site of caspases5.

Mode of action

Caspases cleave the substrate after an Asp residue6.  There are several hundred substrates for caspases.  Initially activation of initiator caspases occurs as a result of an extrinsic or intrinsic death signal6.  Activated initiator caspases cleave effector caspases that in turn cleave the substrate at an Asp residue6.  For example, caspase-8 cleaves the pro-apoptotic protein Bid that gets activated and translocates into the mitochondria where it activates other pro-apoptotic proteins, Bax and Bak thus amplifying the death signal6.

Functions

Caspases such as caspase-1 are involved in the activation of pro-inflammatory cytokines such as Interleukin 1 and interleukin 185,6.  Caspases play an important role in apoptosis. One of the hallmark feature of apoptotic cell death is genomic disassembly and proteolysis5,6.   By cleaving their substartes, caspases inactivate cell cycle progression and DNA repair processes.  They also activate several pro-apoptotic proteins5,6.  In some cases Caspases’ role in aberrant processing events has shown their involvement in neurodegenerative disorders such as Huntington disease and Alzheimer’s disease6.  Some of the final targets of caspases include: nuclear lamins, ICAD/DFF45 (inhibitor of caspase activated DNase or DNA fragmentation factor 45), PARP (poly-ADP ribose polymerase) and PAK2 (P 21-activated kinase 2)6.  Caspases are also implicated in embryonic development and T and B cell differentiation7.

References

1.     Book: Cells by Benjamin L, Lynne C, Vishwanath RL, George P (207), 536-540.

2.     Ellis HM, Horvitz HR (1986). Genetic control of programmed cell death in the nematode C. elegans. Cell, 44(6), 817-29.

3.     Yuan J, Shaham S, Ledoux S, Ellis HM and Horvitz HR (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell 75: 641–652.

4.        Salvesen GS, Riedl SJ (2008). Caspase mechanisms. Adv Exp Med Biol., 615, 13-23.

5.     Prior PF and Salvesen GS (2004). The protein structures that shape caspase activity, specificity,activation and inhibition. Biochem. J., 384, 201–232.

6.     Nicholson DW (1999). Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death and Differentiation, 6, 1028 ± 1042.

7.     Maelfait J, Beyaert R (2008). Non-apoptotic functions of caspase-8. Biochem Pharmacol., 76(11), 1365-73.

Caspase酶對應的底物，Caspases（半胱氨酸天冬氨酸蛋白酶，半胱氨酸依賴性天冬氨酸定向蛋白酶）是一類蛋白酶家族，其功能與凋亡（程序性細胞死亡），壞死和發(fā)燒（炎癥）的過程密切相關。

       什么是胱天蛋白酶？

      胱天蛋白酶（Caspases）是含半胱氨酸的天冬氨酸蛋白水解酶，它們是為細胞凋亡的主要介質。多種受體，例如TNF-α 受體，FasL受體，TLR和死亡受體，以及Bcl-2和凋亡抑制劑（IAP）蛋白家族參與并調節(jié)該caspase依賴性凋亡途徑。一旦Caspase受到上游信號（外部或內在）刺激被激活，即會參與執(zhí)行下游蛋白底物的水解作用，并觸發(fā)一系列事件，導致細胞分解，死亡，吞噬作用和細胞碎片的清除。

      人Caspases酶

      人的Caspases家族基于序列相似性和生物學功能等共性主要可分為三大類：第一類由具有長胱天蛋白酶募集結構域的“炎癥”胱天蛋白酶組成，他們對P4位上的較大的芳香族或疏水性殘基具有親和力。第二類由具有短的前體結構域的“細胞凋亡效應”胱天蛋白酶組成，而第三類由具有長的前提結構域的Pap位置具有亮氨酸或纈氨酸底物親和力的“凋亡引發(fā)劑”胱天蛋白酶組成（表1）。

       表1. 人胱天蛋白酶的功能分類：

       啟動器Caspase和效應器Caspase酶

      根據其在凋亡胱天蛋白酶途徑中的作用，胱天蛋白酶可分為兩類：啟動器和效應器Caspase酶。啟動器和效應器Caspas酶都具有由小亞基和大亞基組成的催化位點，Caspase酶的識別位

      凋亡啟動器Caspase酶，例如caspase-2，-8，-9和-10可以啟動caspase激活級聯反應。Caspase-8對于形成死亡誘導信號復合物（DISC）是必不可少的，并且在激活后，Caspase-8激活下游效應子Caspase（例如Caspase 3）并介導線粒體中細胞色素c的釋放。Caspase-8已被證明對IETD肽序列具有相對較高的底物選擇性。凋亡效應胱天蛋白酶例如Caspase-3，-6和-7雖然不負責啟動級聯途徑，但是當被激活時，它們在級聯的中間和后續(xù)步驟中起著不可或缺的作用。Caspase-3（CPP32 / apopain）是關鍵效應器，因為它放大了來自啟動器Caspase的信號，使用對Caspase-3有選擇性的DEVD肽序列對活化的Caspase-3進行檢測，可以檢測Caspase-3的活性。

       Caspase酶底物和抑制劑

      Caspase底物和抑制劑由兩個關鍵成分組成：Caspase識別序列和信號產生或蛋白酶抑制基序。不同Caspase識別序列不同，一般由三個或四個氨基酸組成（表2）。Caspase酶識別序列的N端通常有乙?；ˋc）或碳苯甲氧基（Z）基團修飾，以增強膜的通透性。對應的Caspase識別特定的肽序列為其酶促反應切割位點，釋放產生信號或抑制信號的基序。Caspase的顯色和熒光底物均以相似的方式起作用，其中底物的信號或顏色強度與蛋白水解活性成正比。

       表2. Caspase的底物及其序列

         Caspase酶的顯色底物

      Caspase的顯色底物是有Caspase識別序列及生色基團組成，常見的生色團有pNA（對硝基苯胺或4-硝基苯胺），可使用酶標儀或分光光度計在405 nm處進行光密度檢測。

       表3. Caspase的顯色底物

       Caspase的熒光底物

      Caspase的熒光底物的結構包含與半胱天冬酶識別相關的熒光團，例如7-氨基-4-甲基香豆素（AMC），7-氨基-4-三氟甲基香豆素（AFC）， Rhodamine 110（R110）或ProRed™620。R110的Caspase底物比基于香豆素的Caspase底物（例如AMC和AFC）更敏感，但由于兩步裂解過程，其動態(tài)范圍更窄。 建議將R110標記的Caspase底物用于終點法測定，而將AMC和AFC標記的 Caspase底物用于動力學測定。

      圖.從左到右，分別是AMC（7-氨基-4-甲基香豆素），AFC（7-氨基-4-三氟甲基香豆素），Rhodamine 110（R110）和ProRed™620的激發(fā)和發(fā)射光譜。

       表4.熒光半胱天冬酶底物。

       注意：

        1.ε=在其最大吸收波長處的摩爾消光系數（單位= cm ^-1M ^-1）。

      2.Φ=水性緩沖液（pH 7.2）中的熒光量子產率。

       Caspase抑制劑

      Caspase抑制劑能與Caspase的活性位點結合并形成可逆或不可逆的連接，通常，Caspase抑制劑的結構由Caspase識別序列，諸如醛（-CHO）或氟甲基酮（-FMK）的官能團組成。具有醛官能團的胱天蛋白酶抑制劑是可逆的，而具有FMK的抑制劑是不可逆的。半胱天冬酶底物和抑制劑都具有較小的細胞毒性作用，因此，它們是研究半胱天冬酶活性的有用工具。

       表5. 可逆和不可逆的Caspase酶抑制劑