(199)enzyme 2011

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Enzypes for MBBS students of first year.I am very thankful to all the colleagues

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  • 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?
  • 上圖 把原核細胞及真核細胞中的基因表現過程作一整理,並且指出整個過程中可供調控的控制點。有許多都是在 DNA 或 RNA 層次的調控,細胞可以藉由開關某基因,而開啟或關閉相對應蛋白質的表現。而此種基因表現的控制方式,是十分複雜的;多是利用某種調節性蛋白質,在基因的前端 ( 調控區 ) 指揮該基因是否能夠被轉錄出 mRNA 。因此細胞內酵素活性的調節,有許多不同的層次,本課程的焦點放在蛋白質生合成之後,對蛋白質進行的種種修飾與控制;至於基因表現層次的調控機制,就是分子生物學的主軸,台大有許多相關課程可以選修。
  • 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 ) 。
  • 酵素 活性區像一個魔術口袋,把反應物放進去後,就可以變成生成物出來。當然我們已經知道,這個口袋可以降低反應中間物的活化能,但它是如何做到的呢? 本圖解提出四個可能的機制。 (1) 過渡狀態分子的構造中,經常都有相當高的局部電荷,而催化口袋內的適當位置上,剛好佈置有可以中和掉此局部高電荷的基團,因而得以穩定過渡狀態。 (2) 在水溶液中,許多離子間的鍵結或反應都會被水分子干擾,因此凹陷的催化口袋可以隔離大多數水分子,使得離子間的反應順利進行。 (3) 在活性區內的胺基酸基團,有些可以因為特別的空間排列,而使得原本反應性低的基團 ( 例如 Ser-OH) ,因為附近其他基團的影響 ( 如 His 可奪取其 H + ) ,而變成具有高反應性的基團 ( 如 Ser-O - ) 。 (4) 活性區通常也是輔 脢 的結合區,輔 脢 分子都帶有強大的電荷基團,可以直接參與反應或者輔助反應進行。
  • S 型 曲線有其特別意義︰ 在 S 曲線上的轉折點 ( 也就是整個線條的中點 ) ,代表 ATCase 的活性在此點之上很快變成活性型,此點之下則保持在非活性型。有點像一個負責開關的關鍵濃度,當基質的濃度到達此點,酵素的活性迅速上升;反之若在此濃度之下,酵素的活性保持著較低的活性。因此,異位 脢 可以說是具有感受環境中基質濃度的能力,藉以調節其活性的大小。 正效應物 ATP 會增加 ATCase 的活性,其動力學的 S 型曲線則變回原來的傳統式 M-M 拋物線。而負效應物 CTP 會降低 ATCase 活性,雖仍保持原來的 S 型曲線,但會往高基質濃度位移,亦即需要更高的基質濃度來維持其正常活性。
  • 上圖 整理出五種酵素的調控方式,其中以抑制劑來抑制酵素的方法與機制已在第四節中介紹過,將不再談。 其餘四種除了胜鍵裂解 (6.1) 是不可逆性的修飾方法外,都是可逆性的調節。 而三種可逆性調節方式當中,只有磷酸化 (6.2) 是共價性修飾,其餘兩種為非共價性的結合,都是利用某種分子與酵素結合而修飾之;其中 cAMP 及 calmodulin (6.3) 是都信息傳導的分子,是把指令由細胞外面傳到裡面的中間人;另外的迴饋控制 (6.4) 則是以細胞內的上下游代謝物質來控制酵素活性。這幾種方法,都同時在生物體中努力地進行細胞內外酵素活性的調控,以便讓細胞達到最有效,而且可以控制自如的生理功能。 近年來,酵素的活性調控方面有很大的進展,尤其是信息傳導的方式極複雜,其五花八門更是令人眼花撩亂。本課程只是一個入門,因此儘量簡化各種所要介紹的主題,通常是以一個比較成熟的實例或機制為故事的主角來說明,點出該主題的最重要主軸;至於深入到何種程度,則通常適可而止,其深度與廣度要靠同學自行去努力。最近台大已經有很多相關課程,深入討論信息傳導,有志者應可挑選適當的課程進一步精研。
  • 異位脢 的最典型例子,就是 aspartate transcarbamoylase (ATCase) 。此酵素催化上圖的反應,所產生的生成物會繼續代謝,最後生成 CTP 。 此 CTP 會回頭與 ATCase 結合,再迴饋抑制其活性 ( 因為 CTP 太多表示不用再繼續此一代謝路徑了 ) 。因為 CTP 與 ATCase 結合在其 R 次體上,而非 C 次體上的活性區,因此是一種道地的異位 脢 。 CTP 之所以能抑制 ATCase 的活性,是因為當 CTP 結合到 R 次體後,會牽動 C 次體的構形,使得 ATCase 由原來活躍的 relaxed form 轉變成較不具活性的 tense form 。
  • 細胞膜上 到底如何傳遞信息? 其方式實在非常多,一般依照 receptor 種類,可整理出三大類主要模式: (1) G-protein-linked Receptor: Receptor 與 G protein 連結後活化之, G protein 本身是 GTP 結合蛋白,與 GTP 結合後可以活化 adenylate cyclase ,後者催化 ATP 成為 cAMP 。 (2) Enzyme-linked Receptor: Receptor 連結著 激 脢 ( 如 tyrosine kinase) ,後者自我磷酸化活化自己,也吸引來一些酵素 ( 如含有 SH2 domain 的磷酸 脢 ) 並且活化之,然後繼續下游的催化路徑。 (3) Ion-channel-linked Receptor: 與細胞膜上的離子主動運輸有關。
  • S 型 曲線有其特別意義︰ 在 S 曲線上的轉折點 ( 也就是整個線條的中點 ) ,代表 ATCase 的活性在此點之上很快變成活性型,此點之下則保持在非活性型。有點像一個負責開關的關鍵濃度,當基質的濃度到達此點,酵素的活性迅速上升;反之若在此濃度之下,酵素的活性保持著較低的活性。因此,異位 脢 可以說是具有感受環境中基質濃度的能力,藉以調節其活性的大小。 正效應物 ATP 會增加 ATCase 的活性,其動力學的 S 型曲線則變回原來的傳統式 M-M 拋物線。而負效應物 CTP 會降低 ATCase 活性,雖仍保持原來的 S 型曲線,但會往高基質濃度位移,亦即需要更高的基質濃度來維持其正常活性。
  • 蛋白質 激 脢 中的 protein kinase A (PKA) 可以對較廣泛的蛋白質進行磷酸化反應,其本身的活性又被 cAMP 所活化。在某些細胞中, PKA 會進入細胞核中,並且磷酸化促進基因轉錄的蛋白質 ( 如上面的 CRE-binding protein, CREB) ,啟動某些基因。 PKA 共有四個次體,含有兩個催化次體 (C) 及兩個調節次體 (R) , R 接受 cAMP 後即釋放出具有活性的催化次體。
  • 酵素 的抑制劑有不同的抑制機制,通常依照抑制劑對酵素的結合方式,可分成兩大類。其一為競爭同一活性區 (competitive) ,可以用提高基質濃度的方法來競爭;另一則是結合在活性區之外的地方,又可分成 non-competitive 及 uncompetitive 兩種。後面兩種抑制方式大致相同,因此有些課本也就不再細分,其差別在於基質的結合,會不會影響抑制劑的結合。雖然這幾種抑制方式,都是可逆反應,但只有 competitive 可以用提高基質的方式來對抗抑制。
  • 競爭性 抑制劑通常都與正常的基質相像,可以與酵素結合,但無法繼續反應,產生生成物;因為都是競爭同一活性區,因此可提高基質來對抗抑制。
  • 這些 抑制機制都可以用酵素動力學來描述,使用雙倒數作圖更可明顯地指出是屬於何種抑制方式。不過,以上三種作圖都是屬於最典型者,很多時候實驗所得到的作圖結果,可能會有混合型態出現,則是較為複雜的抑制機制,或者有其他的干擾因子在內。
  • 抑制劑 在生理或醫藥上,有極重大的作用。例如我們常用的磺胺藥,即所謂的消炎粉,就是一種競爭性抑制劑;磺胺藥分子構造,類似細菌的一種重要代謝物 (PABA) ,因而可與催化 PABA 的酵素結合,造成其抑制。 細胞內有一大群蛋白 脢 ,也在細胞內負責重要的生理功能;而自然界中存在著這些蛋白 脢 的各種抑制劑,有些具生理效果,有些是病理上的致病因子,有些是治病的妙藥;我們將各舉一例,其中可以治療 AIDS 的蛋白 脢 抑制劑,是近年來醫學上的要角之一。
  • 競爭性 抑制劑通常都與正常的基質相像,可以與酵素結合,但無法繼續反應,產生生成物;因為都是競爭同一活性區,因此可提高基質來對抗抑制。
  • 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。
  • 蛋白質 激 脢 中的 protein kinase A (PKA) 可以對較廣泛的蛋白質進行磷酸化反應,其本身的活性又被 cAMP 所活化。在某些細胞中, PKA 會進入細胞核中,並且磷酸化促進基因轉錄的蛋白質 ( 如上面的 CRE-binding protein, CREB) ,啟動某些基因。 PKA 共有四個次體,含有兩個催化次體 (C) 及兩個調節次體 (R) , R 接受 cAMP 後即釋放出具有活性的催化次體。
  • 因為 HIV 蛋白 脢 屬於 aspartyl protease ,其分子中含有兩個 Asp ( 如上圖所示 ) ,因此要使用這類蛋白 脢 的抑制劑來對付 HIV 。問題是,人體內也有相似的 aspartyl protease ,對付 HIV 的抑制劑也對人體有害;因此,要如何找到只對 HIV protease 有抑制作用的藥物? 藥物設計在目前的生物技術產業上,是一支非常重要的研究發展單位;我們可以從人類以及 HIV protease 在分子構造上的差異來下手。 這兩種 proteases 剛好可複習 domain 與 subunit 的概念。 HIV protease 是由兩個相同的次體所組成,是同質二元體,整體四級構造相當對稱;而人體的 Asp protease 則由兩個相似的 domains 所構成,沒有四級構造,但也有兩個 Asp 可夾住水分子,這兩個相似的 domains 可能是由同一基因複製所形成。
  • 抑制劑 在生理或醫藥上,有極重大的作用。例如我們常用的磺胺藥,即所謂的消炎粉,就是一種競爭性抑制劑;磺胺藥分子構造,類似細菌的一種重要代謝物 (PABA) ,因而可與催化 PABA 的酵素結合,造成其抑制。 細胞內有一大群蛋白 脢 ,也在細胞內負責重要的生理功能;而自然界中存在著這些蛋白 脢 的各種抑制劑,有些具生理效果,有些是病理上的致病因子,有些是治病的妙藥;我們將各舉一例,其中可以治療 AIDS 的蛋白 脢 抑制劑,是近年來醫學上的要角之一。
  • 磺胺藥 就是消炎藥,因為其構造類似細菌生長細胞壁所需之 PABA ,會競爭性地抑制利用 PABA 的酵素,因而阻礙細菌的生長,但無法完全殺菌。
  • 細胞膜上 到底如何傳遞信息? 其方式實在非常多,一般依照 receptor 種類,可整理出三大類主要模式: (1) G-protein-linked Receptor: Receptor 與 G protein 連結後活化之, G protein 本身是 GTP 結合蛋白,與 GTP 結合後可以活化 adenylate cyclase ,後者催化 ATP 成為 cAMP 。 (2) Enzyme-linked Receptor: Receptor 連結著 激 脢 ( 如 tyrosine kinase) ,後者自我磷酸化活化自己,也吸引來一些酵素 ( 如含有 SH2 domain 的磷酸 脢 ) 並且活化之,然後繼續下游的催化路徑。 (3) Ion-channel-linked Receptor: 與細胞膜上的離子主動運輸有關。
  • 信息傳導 的說明例,分別有兩個互相對抗的蛋白質家族,共同控制細胞的分裂。 當細胞接受到外界的信息,由細胞膜上的受體接受,然後以 Ras 為主的信息傳導路徑,把信號蛋白 ▲ 傳入細胞核。在細胞核裡,抑制者蛋白原本抓住 E2F 轉錄因子,不讓 E2F 啟動目標基因。當信號蛋白進入核內與抑制者結合,釋出 E2F 與目標基因之啟動子結合,就可啟動細胞分裂。 另一個 P53 家族,有比較保守而謹慎的控制策略, P53 會啟動另一基因,轉錄並轉譯出另一種蛋白質 ( 六角形的援軍蛋白 ) ,進入細胞核與信號蛋白結合,放出抑制者,後者再回去抓住 E2F 轉錄因子,因此又把目標基因關掉,細胞分裂就被中止。 若 P53 發現無法控制該細胞的分裂,有可能惡化成癌細胞時, P53 也會啟動細胞凋亡,把自己的細胞摧毀掉,以免癌化。 這兩個家族好像一個是激進的細胞分裂派 (Ras) ,另一個是保守的控制分裂派 (P53) ,共同制衡維持細胞的正常發展。這兩個家族的蛋白質份子中,若有出現突變而導致失效者,生物個體就很容易得到癌症。
  • 細胞膜上 到底如何傳遞信息? 其方式實在非常多,一般依照 receptor 種類,可整理出三大類主要模式: (1) G-protein-linked Receptor: Receptor 與 G protein 連結後活化之, G protein 本身是 GTP 結合蛋白,與 GTP 結合後可以活化 adenylate cyclase ,後者催化 ATP 成為 cAMP 。 (2) Enzyme-linked Receptor: Receptor 連結著 激 脢 ( 如 tyrosine kinase) ,後者自我磷酸化活化自己,也吸引來一些酵素 ( 如含有 SH2 domain 的磷酸 脢 ) 並且活化之,然後繼續下游的催化路徑。 (3) Ion-channel-linked Receptor: 與細胞膜上的離子主動運輸有關。
  • 細胞 表面的接受體 receptor 非常重要,因為它標明該細胞內的活動為何,以接受正確的外來信息,做出正確的生理反應。以肝糖代謝的細胞為例,可以接受 glucagon 的細胞,就會引發細胞內一連串反應,產生 cAMP 活化 PKA ,此 PKA 接著對幾種酵素進行磷酸化反應,磷酸化的結果使某些酵素活性上升 ( 如 GP kinase, protein phosphatase inhibitor) ,但也使某些酵素活性下降 ( 如 glycogen synthase, protein phosphatase) 。其最後結果,就是使得肝糖的合成降低,增加肝糖降解,以供身體利用葡萄糖產生能量。 上圖的 protein phosphatase 會去除磷酸化 ( 見上圖彎曲向上點線箭頭 ) ,因而降低肝糖磷解 脢 的活性,並且增加肝糖合成 脢 活性,與 glucagon 所要的結果相反,因此在這個細胞中被抑制 ( 雖然也是被磷酸化,但磷酸化後活性降低 ) 。反過來看,此一酵素的 inhibitor 則被活化,進一步控制了 protein phosphatase 的活性。
  • 凡是 可以水解胜肽鍵的酵素,均統稱為蛋白 脢 。蛋白 脢 的種類非常多,我們大致歸納為四大類,均依其催化特性來命名。例如 metal protease 是因為分子中含一金屬離子,此金屬離子不但可維持酵素的正確分子構形,也可以參與催化反應; Ser 及 Cys protease 是因為催化區上含有一個 Ser 或 Cys 胺基酸為主要的催化機團;而 Asp protease 也是因為分子上需要有兩個 Asp 基團,以便抓住水解所需的水分子。 每一類蛋白 脢 家族內,其成員的催化機制都相同,但催化目標的專一性不同;例如 Ser 家族內的 trypsin 嗜好水解鹼性胺基酸,而 chymotrypsin 喜歡較大的芳香基團。 以下將把重點放在 Ser 蛋白 脢 的催化區,看其催化鐵三角如何作用,以及此家族在分子演化上的奇特表現。最有趣的是,這種有效的催化鐵三角,居然也會被其它酵素盜用 ( 或是純屬雷同? ) 。
  • 為了 研究 Ser protease 的催化區,有人把 subtilisin 催化鐵三角上面的胺基酸修改,再看影響修飾後的蛋白 脢 活性大小。結果發現 Ser195 是絕對不可以改變的重要胺基酸, His57 及 Asp102 次之。同時,若把可以穩定過渡狀態分子的 Asn155 改成不具電荷的 Leu ,活性也會下降一千倍。因此,這三個重要胺基酸是不能任意改變,所有 Ser protease 家族成員,都有這三個胺基酸在固定的位置。
  • 把 Ser 家族幾個成員拿出來比較,看催化鐵三角附近胺基酸序列的差異性如何。最重要的 Ser 附近的胺基酸保守性非常高,其它地方也都是半保守性地取代 ( 即極性取代極性、非極性取代非極性 ) 。 成員中有一個 acetylcholinesterase (AchE) 似乎是認養來的, AchE 也有催化鐵三角,但是整體的胺基酸序列並不太像 ( 因此上圖無法列出比較的序列 ) ,好像沒有『血緣』關係。的確如此, AchE 原來並非 Ser 家族的嫡系成員,只是趨同演化出具有與鐵三角類似的催化機制。
  • Acetylcholinesterase 的催化機制與胜肽鍵水解極相似,活性區有高反應性的 Ser 以及 His 和 Asp 的質子接力,也分成 acylation 及 deacylation 兩個階段。
  • 看來 它們是各自發展出類似的催化機制,一個有用的組合不但會被重複使用,還會有趨同演化的情形,天底下大家想的真是都差不多。
  • Acetylcholinesterase (AchE) 是與 Ser protease 家族完全無關的一個酵素,它獨立演化出類似 Ser 催化鐵三角的 Asp-His-Ser 活性區,其作用模式也與 Ser protease 類似,但催化的是水解 acetylcholin 的 ester bond 而非 peptide bond ;事實上,這兩種鍵結的水解方式,也極為相像。這種由不相關的前驅物開始,卻發展出相同的催化機制,稱為 趨同演化 ;而 Ser protease 家族則是由一個相同的始祖開始, 趨異演化 成各個不同的家族成員。 因此,演化現象不但在鉅觀的生物圈發生,同時也在分子層次的微觀世界上演。
  • 肝糖磷解脢 的重要性可由其複雜的調控機制得證,幾乎囊括所有重要的調節方式。因此異位 脢 這種細膩的蛋白質調控方法,當然在肝糖磷解 脢 也有,而且效應物種類繁多。 肝糖磷解 脢 的各種調控機制可以整理成為兩大類,其一為共價性修飾,就是 Ser 14 的磷酸化反應;另一則為非共價性的修飾,正向的活化物只有 AMP 一種,要特別注意並不是 cAMP ;而負面的抑制劑則有許多,例如葡萄糖、 Glc-6-P 、 ATP 與咖啡因。這兩大類效應物真是壁壘分明,例如 AMP 與 ATP 的能量狀況恰好相反,血中的 AMP 濃度高了,表示需要能量,因此活化了肝糖磷解 脢 以便進行磷解反應,取得能量;反之,若血中充滿了 ATP ,則關掉肝糖磷解 脢 活性,不須再降解肝糖。 上圖的右半部需加說明,在磷酸化之後,肝糖磷解 脢 很快由 T 轉換成 R ,因此往下的箭頭較粗。但是即使有磷酸化,當加入負效應物時,也會由 R 轉回 T 而失去活性。
  • 異位脢 的最典型例子,就是 aspartate transcarbamoylase (ATCase) 。此酵素催化上圖的反應,所產生的生成物會繼續代謝,最後生成 CTP 。 此 CTP 會回頭與 ATCase 結合,再迴饋抑制其活性 ( 因為 CTP 太多表示不用再繼續此一代謝路徑了 ) 。因為 CTP 與 ATCase 結合在其 R 次體上,而非 C 次體上的活性區,因此是一種道地的異位 脢 。 CTP 之所以能抑制 ATCase 的活性,是因為當 CTP 結合到 R 次體後,會牽動 C 次體的構形,使得 ATCase 由原來活躍的 relaxed form 轉變成較不具活性的 tense form 。
  • 上圖 整理出五種酵素的調控方式,其中以抑制劑來抑制酵素的方法與機制已在第四節中介紹過,將不再談。 其餘四種除了胜鍵裂解 (6.1) 是不可逆性的修飾方法外,都是可逆性的調節。 而三種可逆性調節方式當中,只有磷酸化 (6.2) 是共價性修飾,其餘兩種為非共價性的結合,都是利用某種分子與酵素結合而修飾之;其中 cAMP 及 calmodulin (6.3) 是都信息傳導的分子,是把指令由細胞外面傳到裡面的中間人;另外的迴饋控制 (6.4) 則是以細胞內的上下游代謝物質來控制酵素活性。這幾種方法,都同時在生物體中努力地進行細胞內外酵素活性的調控,以便讓細胞達到最有效,而且可以控制自如的生理功能。 近年來,酵素的活性調控方面有很大的進展,尤其是信息傳導的方式極複雜,其五花八門更是令人眼花撩亂。本課程只是一個入門,因此儘量簡化各種所要介紹的主題,通常是以一個比較成熟的實例或機制為故事的主角來說明,點出該主題的最重要主軸;至於深入到何種程度,則通常適可而止,其深度與廣度要靠同學自行去努力。最近台大已經有很多相關課程,深入討論信息傳導,有志者應可挑選適當的課程進一步精研。
  • (199)enzyme 2011

    1. 1. ENZYMOLOGY <ul><li>Contribution of Scientists. </li></ul><ul><li>Definitions. </li></ul><ul><li>Mode of Action of Enzymes. </li></ul><ul><li>Factors Influencing Enzyme Activity. </li></ul><ul><li>Enzyme Inhibition. </li></ul><ul><li>Regulation of Enzymes. </li></ul><ul><li>Diagnostic Importance of Enzymes. </li></ul><ul><li>Therapeutic Use of Enzymes. </li></ul>
    2. 2. ENZYMES © 2007 Paul Billiet ODWS BERZELIUS 1835 Starch. Hydrolysis. KUHNE 1878 Enzyme mean yeast. EDWARD BUCHNER Sucrose to Ethanol
    3. 3. ENZYMES © 2007 Paul Billiet ODWS EDWARD BUCHNER N.P.1907 ARTHUR HARDEN N.P. 1929 JAMES SUMNER N.P. 1946 JOHAN NORTHROP N.P. 1946 WILHELM OSTWALD N.P. 1909 WARBURG N.P. 1970
    4. 4. ENZYMES <ul><li>DR.K.S.SODHI,M.D . </li></ul><ul><li>PROFESSOR </li></ul><ul><li>BIO-CHEMISTRY </li></ul><ul><li>MMIMS&R MULLANA AMBALA. </li></ul>© 2007 Paul Billiet ODWS
    5. 5. <ul><li>A GOOD TEACHER IS ALWAYS A GOOD CATALYST IN STUDENTS LIFE. </li></ul>ALWAYS A GOOD CATALYST IN STUDENTS LIFE
    6. 6. DISTRIBUTION OF 17 HORSES <ul><li>OLDMAN AND THREE SONS. </li></ul><ul><li>DISTRIBUTION OF HORSES. </li></ul><ul><li>ELDER ½ </li></ul><ul><li>MIDDLE 1/3 </li></ul><ul><li>LITTLE 1/9 </li></ul>
    7. 7. ENZYMES © 2007 Paul Billiet ODWS EDWARD BUCHNER N.P.1907 ARTHUR HARDEN N.P. 1929 JAMES SUMNER N.P. 1946 JOHAN NORTHROP N.P. 1946 WILHELM OSTWALD N.P. 1909 WARBURG N.P. 1970
    8. 8. Vitro and Vivo Reactions <ul><li>Starch into glucose. Boil for 2 hours in conc Hcl </li></ul><ul><li>For digesting meat need strong acid and long time </li></ul><ul><li>Starch is converted in to glucose in Minutes in the presence of enzymes. </li></ul><ul><li>With in hour at pH 7.4 </li></ul>
    9. 9. DEFINITIONS <ul><li>HOLOENZYMES ( APOENZYMES+CO ENZ.) </li></ul><ul><li>APOENZYMES; SINGLE POLYPEPTIDECHAIN,MORE THAN ONE CHAIN,MULTI-ENZYME COMPLEX. </li></ul><ul><li>Co-ENZYMES : Non Protein (VITAMINS) </li></ul><ul><li>METAL-ACTIVATED ENZYMES.(Zn,Cu,Fe,Mg,K,Ca etc.) </li></ul><ul><li>ZYMASE: Active without modification </li></ul><ul><li>ZYMOGENS : Pro Enzymes eg.Trypsinogen to trypsin </li></ul>
    10. 10. <ul><li>ISO-ENZYMES : Physically distinct perform same function. </li></ul><ul><li>RIBOZYMES: Small ribonuclear particles. </li></ul><ul><li>ENDOENZYMES : Produced in the cell. Function inside the cell. </li></ul><ul><li>EXOENZYMES : Produced inside the cell. Act outside the cell. </li></ul>
    11. 11. <ul><li>METALLO ENZYMES : Contain metal ions as essential component. </li></ul><ul><li>HOUSE KEEPING ENZYMES : Levels of Enzymes can not be controlled. Always present in cell. </li></ul><ul><li>ADAPTIVE ENZYMES : Regulated by genes. Conc.increase or Decrease. </li></ul><ul><li>KEY ENZYMES :Regulatory eg HMG-CO.A </li></ul><ul><li>HYBRID ENZYMES :Produced by genetic fusion. </li></ul>
    12. 12. COFACTORS <ul><li>An additional non-protein molecule that is needed by some enzymes to help the reaction </li></ul><ul><li>Tightly bound cofactors are called prosthetic groups </li></ul><ul><li>Cofactors that are bound and released easily are called coenzymes </li></ul><ul><li>Many vitamins are coenzymes </li></ul>Nitrogenase enzyme with Fe, Mo and ADP cofactors
    13. 13. CO-ENZYMES <ul><li>Essential for Biological activity. </li></ul><ul><li>Low molecular weight, Organic in nature </li></ul><ul><li>Non protein in nature. </li></ul><ul><li>.Combine loosly with Enzyme &separate later. </li></ul><ul><li>Thermostable. </li></ul><ul><li>Help in group transfer. </li></ul><ul><li>Bind to apoenzymes. </li></ul><ul><li>Eg.NAD, NADP, FMN, FAD, Biotin, Lipoic Acid, Pyridoxal Phosphate,etc. (Vitamins) </li></ul><ul><li>Co-enzyme separate from apo-Enz after reaction. </li></ul><ul><li>Can be separated by Dialysis. </li></ul>
    14. 14. <ul><li>Co-Enzymes can be divided into two groups. </li></ul><ul><li>A.Oxidoreductases.NADH.NADPH,FAD. </li></ul><ul><li>B. Transfer Groups. </li></ul><ul><li>Thiamine-Hydroxyl group. </li></ul><ul><li>Pyridoxal phosphate-Amino group </li></ul><ul><li>Tetrahydrofolate-one Carbon </li></ul><ul><li>Biotin-Carbon dioxide. </li></ul>
    15. 15. TABULAR FORM SHOWING CO.E
    16. 16. Enzyme structure <ul><li>Enzymes are proteins </li></ul><ul><li>They have a globular shape </li></ul><ul><li>A complex 3-D structure </li></ul>© 2007 Paul Billiet ODWS Human pancreatic amylase
    17. 17. STRUCTURE <ul><li>1.MONOMERIC: Single Peptide. </li></ul><ul><li>2.OLIGOMERIC: Many peptide Chains. </li></ul><ul><li>3.Multienzyme Complex: </li></ul><ul><li>Fatty Acid Synthase </li></ul><ul><li>LDH Complex. </li></ul><ul><li>Prostaglandin Synthase Complex. </li></ul>
    18. 18. ENZYMES UNITS <ul><li>KINGARMSTRONG. </li></ul><ul><li>SOMOGY. </li></ul><ul><li>REITMAN FRANKEL. </li></ul><ul><li>SPECTROPHOTOMETRIC. </li></ul><ul><li>KATAL. </li></ul><ul><li>INTERNATIONAL UNIT. </li></ul>
    19. 19. ENZYMEZS ESTIMATED FROM: <ul><li>WHOLE BLOOD, SERUM, PLASMA. </li></ul><ul><li>RED BLOOD CELLS. </li></ul><ul><li>C.S.F. </li></ul><ul><li>URINE. </li></ul><ul><li>SWEAT. </li></ul><ul><li>SALIVA. </li></ul><ul><li>SEMEN. </li></ul><ul><li>AMNIOTIC FLUID. </li></ul><ul><li>Tears. </li></ul>
    20. 20. PLASMA ENZYMES <ul><li>FUNCTIONAL PLAMSMA ENZYMES. eg. LIPOPROTEIN LIPASE, BLOOD CLOT DISSOLVING ENZYMES etc. </li></ul><ul><li>NON FUNCTIONAL PLASMA ENZYMES. eg: SGOT, SGPT,AMYLASE,CPK,LDH,LIPASE,ACID-PHOSPHATASE,ALKALINE PHOS., CERULOPLASMIN etc. </li></ul>
    21. 21. NATURE OF ENZYMES <ul><li>Soluble, Colloidal, Organic Catalysts </li></ul><ul><li>Formed by Living Cells ,Specific in action, Protein In Nature ,Inactive at Zero degree centigrade ,Destroyed by moist heat at 100 degree centigrade (Heat Labile), Huge in size, small Active Site, Used for Treatment. </li></ul>
    22. 22. DIFFERENCE <ul><li>BIO-CATALYST : </li></ul><ul><li>Enzymes, protein in nature except ribozymes, More specific, more efficient and slight change in structure alter its action. </li></ul><ul><li>CATALYST: </li></ul><ul><li>Inorganic, less sp., less efficient and no change in structure. </li></ul>
    23. 23. THE ENZYMES SPEAK <ul><li>“ WE ARE THE CATALYSTS OF THE LIVING WORLD? PROTEIN IN NATURE, AND IN ACTION. SPECIFIC, RAPID AND ACCURATE; HUGE IN SIZE BUT WITH SMALL ACTIVE CENTRE; HIGHLY EXPLOITED FOR DISEASE DIAGNOSIS IN LAB CENTRES AND ALSO USED FOR TREATMENT.’’ </li></ul>
    24. 24. TISSUES BRAIN,HEART,LIVER,KIDNEY,MUSCLE MUSCLE -> ← HEART -> LIVER ← STOMACH BRAIN ← KIDNEY ← INTESTINE
    25. 26. COMPARTMENTATION <ul><li>MITOCHONDRIA: Enzymes of: E.T.C, TCA Cycle, Beta Oxidation, Urea Cycle, </li></ul><ul><li>Pyruvate to Acetyle Co-A. </li></ul><ul><li>CYTOSOL: Glycolysis, HMP Shunt, Fatty Acid Synthesis, Glucogenesis and Glycogenolysis. </li></ul><ul><li>NUCLEUS: DNA Synthesis, RNA Synthesis and Histones etc. </li></ul><ul><li>LYSOSOMES : </li></ul>
    26. 28. FUNCTIONS OF ENZYMES <ul><li>1. Catalyse thousands of reactions. </li></ul><ul><li>2. Digestive Enzymes help in Digestion. </li></ul><ul><li>3. Lysosomal Enzymes destroy in cell. </li></ul><ul><li>4. Lysozymes are bacteriocidal, local immunity. (TEARS) </li></ul><ul><li>4. Detergents </li></ul><ul><li>5. Textile. </li></ul><ul><li>6. Leather Industry. </li></ul>
    27. 31. What is a Ribozyme? 1) Enzyme 2) Ribonucleic Acid NOT PROTEIN 1989 Nobel Prize In Chemistry Sid Altman Tom Cech
    28. 32. RIBOZYMES <ul><li>Small ribonuclear particles. </li></ul><ul><li>Contain rRNA. </li></ul><ul><li>Highly substrate specific. </li></ul><ul><li>Used in Intron splicing,pre RNA to RNA Peptidyl Transferase. </li></ul><ul><li>Many ribozymes have hair-pin or hammer head shaped active centre &require Divalent Mg++ </li></ul><ul><li>Catalyse reaction on phosphpdiester bonds of other RNA </li></ul>
    29. 33. Ribozymes Have following Drawbacks. <ul><li>Not as efficient as protein catalysts( In RNA there are 4 nucleotides, in amino acid are 20 in number. </li></ul><ul><li>Act once only in chemical event,protein enzymes are reused several times. </li></ul><ul><li>Rate of catalytic activity is slower. </li></ul><ul><li>Synthatic Ribozymes are having better catalytic activity(Cleave infectious Virus) </li></ul><ul><li>Used in Gene therapy. </li></ul>
    30. 34. ABZYMES <ul><li>Artificially synthasized catalytic antibodies against Enz. Sub. Complex in transition state of reaction. CATMAB (Catalytic Monoclonal Antibody). </li></ul><ul><li>Sometimes natural abzymes are found in blood,eg.antivasoactive intestinal peptide autoantibodies. </li></ul><ul><li>Useful in diseases viz.abzyme against gp120 envelop protein of HIV may prevent virus entry in to the host cell. </li></ul>
    31. 35. Structure : As with proteins, we consider... Primary: GGCCGAACUGGUA Secondary: Tertiary:
    32. 36. Secondary Structure Watson-Crick Base Pairing Helix Formation B-DNA Small pore along helical axis “ Rungs” stack obliquely to axis A-DNA RNA RNA usually assumes A-form helices…
    33. 37. Secondary Structure Conserved base-pairing interactions result in... <ul><li>Three “stem” regions </li></ul><ul><li>Uridine-containing turn </li></ul><ul><li>An “augmenting helix” </li></ul><ul><li>joining stems II and III </li></ul>
    34. 38. Ribozyme vs. tRNA Phe folding
    35. 39. Tertiary Structure
    36. 40. The Future of Ribozymes In Vitro Molecular Evolution of RNA High Throughput Screening Ribozyme-Based Therapies +
    37. 41. In Clinical Trial... HIV Gene Therapy... Bone Marrow Sample Treat Stem Cells with Retroviral Vector Re-Implant Treated Cells Encodes Gene for anti-HIV Ribozyme
    38. 42. ACTIVE SITE OF RIBONUCLEASES <ul><li>It lies in a hydrophobic cleft. </li></ul><ul><li>7 th Lysine 41 st Lysine on one side and 12 th Histidine and 119Histidine on the opposite side.(URIDYLIC ACID) </li></ul><ul><li>Peptidyl transferase (chain Elongation) </li></ul><ul><li>Removal of Introns. </li></ul>
    39. 43. The Substrate <ul><li>The substrate of an enzyme are the reactants that are activated by the enzyme </li></ul><ul><li>Enzymes are specific to their substrates </li></ul><ul><li>The specificity is determined by the active site </li></ul>© 2007 Paul Billiet ODWS
    40. 44. PRODUCT <ul><li>Substrate in the presence of Enzyme is converted in to product. </li></ul><ul><li>The reaction can be Reversible or Ir-reversible. </li></ul><ul><li>The increase in product concentration can cause inhibition and stop the reaction in the forwaed direction. </li></ul>
    41. 45. ABBREVIATIONS <ul><li>ENZYME [E] </li></ul><ul><li>SUBSTRATE [S] </li></ul><ul><li>PRODUCT [P] </li></ul><ul><li>Enz. Sub. Complex [ES] </li></ul><ul><li>INHIBITOR [I] </li></ul><ul><li>Enz.+Inh. Complex [ EI ] </li></ul><ul><li>Enz.+Sub.+Inh. [ESI] </li></ul>
    42. 46. Enzyme Stabilizes Transition State S P ES ES T EP S T Energy change Energy required (no catalysis) Energy decreases (under catalysis) Sub.(S) Prod. (P)Enz(E) T = Transition state V=rate of change of S to P/mt. Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166 Reaction direction
    43. 48. Control Points of Gene Regulation Prokaryotics Post-translational control Eukaryotics Juang RH (2004) BCbasics DNA ribosome mRNA proteins proteins cap 5’ 3’ tail mature mRNA DNA 5’ 3’ process mRNA Translation Activity Proteolysis Transcription RNA Processing RNA Transport RNA Degradation
    44. 50. ACTIVE SITE OF ENZYME <ul><li>Chymotrypsin His(57)Asp(102)Ser(195) </li></ul><ul><li>Trypsin Histidine,Serine </li></ul><ul><li>Phosphoglucomutase Serine </li></ul><ul><li>Carboxypeptidase Histidine,Arginine,tyrosine </li></ul><ul><li>Aldolase Lysine </li></ul>
    45. 51. Active Site Avoids the Influence of Water Preventing the influence of water sustains the formation of stable ionic bonds - +
    46. 52. Active Site Is a Deep Buried Pocket Why energy required to reach transition state is lower in the active site? It is a magic pocket (1) Stabilizes transition (2) Expels water (3) Reactive groups (4) Coenzyme helps (2) (3) (4) (1) CoE + - Juang RH (2004) BCbasics
    47. 53. <ul><li>The active site : </li></ul><ul><li>Is a region within an enzyme that fits the shape of molecules called substrates . </li></ul><ul><li>Contains amino acid R groups that align and bind the substrate. </li></ul><ul><li>Releases products when the reaction is complete. </li></ul>Active Site
    48. 54. ACTIVE SITE <ul><li>Generally the active site is situated on the cleft of the Enzyme. </li></ul><ul><li>Binding of substrate to active site dependends upon the presence of sp. Groups or atoms at active site. </li></ul><ul><li>During binding these groups,realign themselves so as to fit the substrate. </li></ul><ul><li>The substrate bind to active site by non co-valent bonds.(Hddrophobic in nature) </li></ul><ul><li>Amino acid that make or break bonds called catalytic group. </li></ul>
    49. 55. ACTIVE SITE
    50. 58. MECHANISM OF ACTION <ul><li>INDUCE FIT MODEL.(KOSHLAND’S) </li></ul><ul><li>LOCK AND KEY MODEL. (FISHER’S TEMPLATE THEORY) </li></ul>
    51. 59. The Induced Fit Hypothesis <ul><li>Some proteins can change their shape (conformation) </li></ul><ul><li>When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation </li></ul><ul><li>T he active site is then moulded into a precise conformation </li></ul><ul><li>Making the chemical environment suitable for the reaction </li></ul><ul><li>The bonds of the substrate are stretched to make the reaction easier (lowers activation energy) </li></ul>© 2007 Paul Billiet ODWS
    52. 60. Induced-fit Model <ul><li>In the induced-fit model of enzyme action: </li></ul><ul><li>The active site is flexible, not rigid. </li></ul><ul><li>The shapes of the enzyme, active site, and substrate adjust to maximum the fit, which improves catalysis. </li></ul><ul><li>There is a greater range of substrate specificity. </li></ul>
    53. 61. The Lock and Key Hypothesis <ul><li>Fit between the substrate and the active site of the enzyme is exact </li></ul><ul><li>Like a key fits into a lock very precisely </li></ul><ul><li>The key is analogous to the enzyme and the substrate analogous to the lock. </li></ul><ul><li>Temporary structure called the enzyme-substrate complex formed </li></ul><ul><li>Products have a different shape from the substrate </li></ul><ul><li>Once formed, they are released from the active site </li></ul><ul><li>Leaving it free to become attached to another substrate </li></ul>© 2007 Paul Billiet ODWS
    54. 62. Lock-and-Key Model <ul><li>In the lock-and-key model of enzyme action: </li></ul><ul><li>The active site has a rigid shape. </li></ul><ul><li>Only substrates with the matching shape can fit. </li></ul><ul><li>The substrate is a key that fits the lock of the active site. </li></ul><ul><li>Rigid structure could not explain flexibility shown by enzymes </li></ul>
    55. 64. CLASSIFICATION <ul><li>1. O XIDO-REDUCTASE.transfer of hydrogen or addition of oxygen.Eg.LDH </li></ul><ul><li>2. T RANSFERASE.Eg.Aminotransferase. </li></ul><ul><li>Hexokinase. </li></ul><ul><li>3. H YDROLASE.Cleave bond adding water </li></ul><ul><li>Eg. Acetyl choline estrase. </li></ul><ul><li>4. L YASE.Cleave without adding water (Aldolase) </li></ul><ul><li>5. I SOMERASE. </li></ul><ul><li>6. L IGASE.Acetyl co-A carboxylase,Glu.Synthatase,PRPP Synthatase. </li></ul>
    56. 65. FACTORS AFFECTING ENZYME <ul><li>1.SUBSTRATE CONCENTRATION. </li></ul><ul><li>2.ENZYME CONCENTRATION. </li></ul><ul><li>3.TEMPERATURE. </li></ul><ul><li>4.pH. </li></ul><ul><li>5.EFFECT OF PRODUCT CONC. </li></ul><ul><li>6.PRESENCE OF ACTIVATORS </li></ul><ul><li>7.INHIBITORS. </li></ul><ul><li>8.EFFECT OF TIME. </li></ul>
    57. 66. FACTORS ……………….. <ul><li>9.EFFECT OF CLOSE CONTCT. </li></ul><ul><li>10.OXIDATION OF ADD.GROUPS. </li></ul><ul><li>11.EFFECT OF LIGHT. </li></ul><ul><li>12.EFFECTS OF RADIATIONS. </li></ul><ul><li>13.PRESENCE OF REPRESSOR </li></ul><ul><li>DEPRESSOR </li></ul><ul><li>14. ANTIZYMES. </li></ul>
    58. 67. Substrate concentration: Non-enzymic reactions <ul><li>The increase in velocity is proportional to the substrate concentration </li></ul>Reaction velocity Substrate concentration
    59. 68. Substrate Concentration <ul><li>The rate of reaction increases as substrate concentration increases (at constant enzyme concentration). </li></ul><ul><li>Maximum activity occurs when the enzyme is saturated. </li></ul>
    60. 69. Substrate concentration: Enzymic reactions when[ s] conc. Is increased velocity increases in the initial phase (Vmax.),but flatten afterward. <ul><li>Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. </li></ul><ul><li>If you alter the concentration of the enzyme then V max will change too. </li></ul>© 2007 Paul Billiet ODWS Reaction velocity Substrate concentration V max
    61. 70. Salient Features of Km <ul><li>Km is sub. Conc.at ½ the max. velocity </li></ul><ul><li>It denotes that 50% of Enzyme mol.are bound with sub.at particular sub. Conc. </li></ul><ul><li>Km is independent of Enzyme conc.If Enz. Conc. Is doubled, the Vmax will be double but km will remain same. </li></ul><ul><li>Km is signature of Enzyme. </li></ul><ul><li>Affinity of Enz. Towards its substrate is inversely related to the dissociation constant(smaller the dissociation greater the affinity. </li></ul><ul><li>Km denotes affinity of enzme for substrate.lesser the Km more the affinity. </li></ul>
    62. 71. Enzyme Concentration <ul><li>The rate of reaction increases as enzyme concentration increases (at constant substrate concentration). </li></ul><ul><li>At higher enzyme concentrations, more substrate binds with enzyme. </li></ul><ul><li>End point reaction. </li></ul>
    63. 72. The effect of temperature <ul><li>For most enzymes the optimum temperature is about 30°C </li></ul><ul><li>Many are a lot lower, cold water fish will die at 30°C because their enzymes denature </li></ul><ul><li>A few bacteria have enzymes that can withstand very high temperatures up to 100°C </li></ul><ul><li>Most enzymes however are fully denatured at 70°C </li></ul>© 2007 Paul Billiet ODWS
    64. 73. <ul><li>Enzymes : </li></ul><ul><li>Are most active at an optimum temperature (usually 37°C in humans). </li></ul><ul><li>Show little activity at low temperatures. </li></ul><ul><li>Lose activity at high temperatures as denaturation occurs. </li></ul>Temperature and Enzyme Action
    65. 74. The effect of temperature Temperature / °C Enzyme activity 0 10 20 30 40 50 Q10 Denaturation
    66. 75. The effect of temperature <ul><li>Q10 ( the temperature coefficient ) = the increase in reaction rate with a 10°C rise in temperature. </li></ul><ul><li>For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature) </li></ul><ul><li>Enzyme-controlled reactions follow this rule as they are chemical reactions </li></ul><ul><li>BUT at high temperatures proteins denature </li></ul><ul><li>The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation. </li></ul>
    67. 76. The effect of pH <ul><li>Extreme pH levels will produce denaturation </li></ul><ul><li>The structure of the enzyme is changed </li></ul><ul><li>The active site is distorted and the substrate molecules will no longer fit in it </li></ul><ul><li>At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur </li></ul><ul><li>This change in ionisation will affect the binding of the substrate with the active site. </li></ul>© 2007 Paul Billiet ODWS
    68. 77. The effect of pH Optimum pH values © 2007 Paul Billiet ODWS Enzyme activity Trypsin Pepsin pH 1 3 5 7 9 11
    69. 78. <ul><li>Enzymes : </li></ul><ul><li>Are most active at optimum pH. </li></ul><ul><li>Contain R groups of amino acids with proper charges at optimum pH. </li></ul><ul><li>Lose activity in low or high pH as tertiary structure is disrupted. </li></ul>pH and Enzyme Action
    70. 79. Optimum pH Values <ul><li>Most enzymes of the body have an optimum pH of about 7.4. </li></ul><ul><li>In certain organs, enzymes operate at lower and higher optimum pH values. </li></ul>
    71. 81. ENZYME ACTIVATION BY INORGANIC IONS <ul><li>In the presence some inorganic ions some enzymes show higher activity eg.Chloride ion activate salivary amylase,Ca. activates lipases. </li></ul><ul><li>Proenzymes in to enzymes. </li></ul><ul><li>Coagulatio factors are seen in blood as zymogen. </li></ul><ul><li>Compliment cascade,these activities needed occasionly. </li></ul>
    72. 82. Enzyme Inhibition <ul><li>Competitive Inhibtion. </li></ul><ul><li>Non-Competitive Inhibition. </li></ul><ul><li>Un-competitive Inhibition. </li></ul><ul><li>Suicide Inhibition. </li></ul><ul><li>Allosteric Inhibition </li></ul><ul><li>Key Enzymes </li></ul><ul><li>Feedback Inhibition. </li></ul><ul><li>Inducors.Glucokinase is induced by Insulin. </li></ul><ul><li>Repression (Heme is reprossor of ALA Synthase. </li></ul>
    73. 84. Sigmoidal Curve Effect Sigmoidal curve Exaggeration of sigmoidal curve yields a drastic zigzag line that shows the On/Off point clearly Positive effector (ATP) brings sigmoidal curve back to hyperbolic Negative effector (CTP) keeps Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity Noncooperative (Hyperbolic) Cooperative (Sigmoidal) v o [Substrate] Off On Juang RH (2004) BCbasics CTP ATP v o
    74. 85. EFFECT OF CONC.PRODUCT <ul><li>At Equilibrium as per law of mass action,the reaction rate is slowed down,it can slow,stopped or reversed. </li></ul><ul><li>A —E1— B —E2—≠— C —E3— D . </li></ul><ul><li>Increase in conc. Of D will cause feed back Inhibition. </li></ul>
    75. 87. INDUCTION <ul><li>Induction is effected through the process of derepression. </li></ul><ul><li>The inducer will relieve the repression on the operator site. </li></ul><ul><li>In the absence of glucose,the enzymes of Lactose metabolism will increase thousand times. </li></ul><ul><li>Insulin is Inducer of Hexokinase Enzyme. </li></ul><ul><li>Barbiturates induce ALA Synthase. </li></ul>
    76. 88. REPRESSION <ul><li>Inhibition and repression reduce the Enzyme Velocity. </li></ul><ul><li>In case of Inhibition the Inhibitor act directly on the Enzyme. </li></ul><ul><li>Repressor acts at the gene level,effect is noticed after a lag period of Hours or Days. </li></ul>
    77. 89. CO VALENT MODIFICATION <ul><li>Activity of Enzyme can be increased or decreasd by co-valent modifications Eg.Either addition of group or Removal of group </li></ul><ul><li>Zymogen activation by partial proteolysis is an Eg. Of co-valent modification </li></ul>
    78. 90. ADP RIBOSYLATION <ul><li>It is a type of co-valent modification. </li></ul><ul><li>ADP-Ribose from NAD is added to enzyme/Protein. </li></ul><ul><li>ADP Ribosylation of Alfa Sub unit of G Protein leads to Inhibition of GTPase activity;hence G protein remains active. </li></ul><ul><li>Cholera toxin & Pertussive toxin act through ADP-Ribosylation. </li></ul><ul><li>ADP Ribosylation of Glyeraldehyde 3P-Dehydrosense,result in inhibition of glycolysis. </li></ul>
    79. 91. STABILIZATION <ul><li>Enzyme molecules undergo usual wear & tear finally get degraded.Enzymes having thio (SH) groups eg Papian,Succinate dehydrogenase are stablized by glutathione. (G-SH). </li></ul><ul><li>Phosphofructokinase is stablized by growth hormone. </li></ul>
    80. 92. Regulation of Enzyme Activity P R R + proteolysis phosphorylation cAMP or calmodulin or regulator effector (+) Juang RH (2004) BCbasics Regulatory subunit or inhibitor P (-) Inhibitor Proteolysis Phosophorylation Signal transduction Feedback regulation
    81. 93. REGULATION OF ENZYMES <ul><li>‘’ The action of enzymes can be activated or inhibited so that the rate of enzyme productin responds to the physiologcal need of the cell done to achieve cellular economy’’ </li></ul>
    82. 94. <ul><li>1. Allosteric Regulation. </li></ul><ul><li>2. Activation of Latent Enzyme. </li></ul><ul><li>3. Comprtmentation of Enzymes of different Pathways. </li></ul><ul><li>4. Control of Enzyme Synthesis. </li></ul><ul><li>5. Enzyme Degradation. </li></ul>
    83. 95. REGULATION OF ENZYMES <ul><li>Control of metabolic pathway occour through modification of Enzyme activity. </li></ul><ul><li>One or more key enzyme on the pathway. Can be involved in regulation. </li></ul><ul><li>One enzyme that regulate is called rate limiting enzyme or key enzyme of that path –way.Usually this is first enzyme. </li></ul><ul><li>This enzymes activity change in quantity of enzyme present orIntrinsic catalytic efficiency of enz. Molecule. </li></ul>
    84. 96. CHANGE IN ENZYME QUANTITY <ul><li>Absolute Quantity of Enz.is determined by balance (Rate of Synthesis&Degradation) </li></ul><ul><li>Enzyme whose synthesis is increased by inducer are called inducible enz.Eg. Tryptophan pyrolase,Tyrosine Transaminase and HMG co-A Reductase. </li></ul><ul><li>Enz. Whose conc. Always maintained at particular level,independent of Inducor are called constitutive enz. Eg. Hexokinase. </li></ul><ul><li>Sometimes accumulation of metabolite inhibit its own synthesis.These are called repressor. </li></ul>
    85. 97. CHANGE IN CATALYTIC EFFICIENCY OF ENZYME <ul><li>Catalytic effeciency is regulated is modulated by </li></ul><ul><li>A. Allosteric Regulation. </li></ul><ul><li>B. Covalent modification . </li></ul><ul><li>A. ALLOSTERIC REGULATION: Here the site is different from substrate binding site, this site is called ALLOSTERIC SITE. </li></ul><ul><li>Low molecular wt. substances bind at site other than catalytic site,these are called ALLOSTERIC MODULATORS.Location is called allosteric site. </li></ul>
    86. 99. A.Activator A.Inhibitor . <ul><li>Allosteric Activator . </li></ul><ul><li>Hexokinase: ADP </li></ul><ul><li>Isocit.Dehydr. ADP </li></ul><ul><li>Glu.Dehy. ADP </li></ul><ul><li>Pyruvate Carboxylase </li></ul><ul><li>Acetyl CoA </li></ul><ul><li>Allosteric Inhibitor . </li></ul><ul><li>Glucose-6-P,ATP </li></ul><ul><li>Glucose-6-P,ATP </li></ul><ul><li>ATP, NADH. </li></ul><ul><li>ADP </li></ul>
    87. 100. <ul><li>HOMOTROPIC EFFECT : If the effector </li></ul><ul><li>Substace is substrate itself it is called homotropic effect. </li></ul><ul><li>HETEROTROPIC EFFECT : Effector molecule is a substance other than substrate. </li></ul><ul><li>SECOND MESSENGER:Binding of many hormones to their surface receptor induce a change in enzyme catalysed reaction by inducing the release of allosteric effector.These effector substances are called as 2 nd messenger </li></ul><ul><li>Hormone is first messenger. Cont…… </li></ul>
    88. 101. <ul><li>Examples of Second messengers are cAMP,cGMP and calcium etc. </li></ul><ul><li>These can change the enzyme conformation that may alter either Km or Vmax. </li></ul><ul><li>Based on this effect they are classified in to two classes. </li></ul><ul><li>1.K-class:Alter Km not Vmax. </li></ul><ul><li>2.V-class:Alter Vmax not Km. </li></ul>
    89. 102. <ul><ul><li>CONFERMATIONAL CHANGES IN ALLOSTERIC ENZYMES . </li></ul></ul><ul><li>Most of Enzymes are oligomeric, binding of effector moecule at the allosteric site brings a chage in the active site of enzyme leading to inhibition or activation. </li></ul><ul><li>Allosteric Enzyme exhist in two states. </li></ul><ul><li>A. Tense (T) </li></ul><ul><li>B. Relaxed (R ) </li></ul><ul><li>Both are in equlibrium. </li></ul>
    90. 103. CCC Allosteric Enzyme ATCase + Active relaxed form Inactive tense form ATCase R R R R R R CCC Catalytic subunits Catalytic subunits Regulatory subunits Aspartate Carbamoyl phosphate Carbamoyl aspartate Juang RH (2004) BCbasics Quaternary structure COO - CH 2 HN-C-COO - H H - - - - O H 2 N-C-O-PO 3 2- = O H 2 N-C- = COO - CH 2 N-C-COO - H H - - - - ATP CTP Nucleic acid metabolism Feedback inhibition CTP CTP CTP CTP CTP CTP
    91. 104. EXAMPLE OF 2 nd MESSENGER <ul><li>GLYCOGEN BREAKDOWN . </li></ul><ul><li>GLYCOGEN SYNTHESIS. </li></ul>
    92. 105. The Reception and Transduction of Signals -GDP +GTP Adenylate cyclase   Glycogen Synthase active Insulin kinase Glucagon A The third group: Ion-channel-linked Receptor Gilman, Rodbell (1994) Glycogen breakdown Juang RH (2007) BCbasics SH2 domain  G protein   GDP   + Signal  GDP  GTP  GTP + Signal Activation P Protein Phosphatase Glycogen Synthase P P P P P G-protein-linked Receptor Enzyme-linked Receptor Glycogen
    93. 106. Sigmoidal Curve Effect Sigmoidal curve Exaggeration of sigmoidal curve yields a drastic zigzag line that shows the On/Off point clearly Positive effector (ATP) brings sigmoidal curve back to hyperbolic Negative effector (CTP) keeps Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity Noncooperative (Hyperbolic) Cooperative (Sigmoidal) v o [Substrate] Off On Juang RH (2004) BCbasics CTP ATP v o
    94. 107. FEED BACK INHIBITION <ul><li>Enzyme is inhibited by end product of reaction. </li></ul><ul><li>A-B-C-D-E-F……….P. </li></ul><ul><li>P product will inhibit the enzyme which converts A in to B. </li></ul>
    95. 109. COVALENT MODIFICATIONS <ul><li>Two well known processes </li></ul><ul><li>A. PHOSPHORILATION. </li></ul><ul><li>B. PARTIAL PROTEOLYSIS. </li></ul><ul><li>A. Phosphorilation-dephosphorilation:many enzymes are regulated by ATP dependent phosphorilation.Eg. Of Serine,Threonine,and tyrosine,catalysed by protein kinases. </li></ul>
    96. 110. cAMP Controls Activity of Protein Kinase A A A A A cAMP Active kinase CREB CREB Nucleus Activation Gene expression ON DNA Alberts et al (2002) Molecular Biology of the Cell (4e) p. 857, 858 R C R C R R A A A A C C Regulatory subunits Catalytic subunits C P
    97. 111. PARTIAL PROTEOLYSIS <ul><li>Some enzymes are secreted as inactive precursors called Proenzymes or Zymogens. </li></ul><ul><li>This convertion takes place as a selective proteolysis. </li></ul><ul><li>It is ir-reversible process </li></ul><ul><li>Pepsinogen to pepsin </li></ul><ul><li>Trypsinogen to trypsin. </li></ul>
    98. 112. MICHAELIS CONSTANT (Km) <ul><li>It is defined as the conc. Of the substrate at which the reaction velocity is half of the maximum velocity. </li></ul><ul><li>Km is independent of enzyme conc. </li></ul><ul><li>If an enzyme has a small value of Km, it achieves maximal catalytic efficency at low substrate conc. </li></ul><ul><li>SIGNIFICANCE </li></ul><ul><li>Glucokinase has high Km is low affinity for glucose </li></ul>
    99. 113. <ul><li>Hexokinase have low KM High affinity for Glucose ie glucose will provide to the vital organs even at low glucose levels. </li></ul><ul><li>Lab. Significance: The sub. Conc. Kept at saturation point at least 10 times the Km so that reaction proceeds to completion. </li></ul><ul><li>Clinical Significance: The Km value for the given enzyme may differ from person to person and explains various response to drugs/chemicals. </li></ul>
    100. 114. INHIBITORS
    101. 115. Inhibitors <ul><li>Inhibitors are chemicals that reduce the rate of enzymic reactions. </li></ul><ul><li>The are usually specific and they work at low concentrations. </li></ul><ul><li>They block the enzyme but they do not usually destroy it. </li></ul><ul><li>Many drugs and poisons are inhibitors of enzymes in the nervous system. </li></ul>© 2007 Paul Billiet ODWS
    102. 116. The effect of enzyme inhibition <ul><li>Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly </li></ul><ul><li>Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase </li></ul>© 2008 Paul Billiet ODWS
    103. 117. The effect of enzyme inhibition <ul><li>Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. </li></ul><ul><li>There are two categories </li></ul>© 2008 Paul Billiet ODWS
    104. 118. The effect of enzyme inhibition <ul><li>Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site </li></ul><ul><li>Examples </li></ul><ul><li>Cyanide combines with the Iron in the enzymes cytochrome oxidase </li></ul><ul><li>Heavy metals, Ag or Hg , combine with –SH groups. </li></ul><ul><li>These can be removed by using a chelating agent such as EDTA </li></ul>© 2008 Paul Billiet ODWS
    105. 119. Applications of inhibitors <ul><li>Negative feedback : end point or end product inhibition </li></ul><ul><li>Poisons snake bite, plant alkaloids and nerve gases </li></ul><ul><li>Medicine antibiotics, sulphonamides, sedatives and stimulants </li></ul>© 2008 Paul Billiet ODWS
    106. 120. <ul><li>Cell processes (e.g. respiration or photosynthesis) consist of series of pathways controlled by enzymes </li></ul><ul><li>A B C D E F </li></ul>Enzyme pathways Each step is controlled by a different enzyme ( e A , e B , e C etc) This is possible because of enzyme specificity © 2008 Paul Billiet ODWS e F e D e C e A e B
    107. 121. End point inhibition <ul><li>The first step (controlled by e A ) is often controlled by the end product ( F ) </li></ul><ul><li>Therefore negative feedback is possible </li></ul><ul><li>A B C D E F </li></ul><ul><li>The end products are controlling their own rate of production </li></ul><ul><li>There is no build up of intermediates (B, C, D and E) </li></ul>e F e D e C e A e B © 2008 Paul Billiet ODWS Inhibition
    108. 122. ATP is the end point <ul><li>This reaction lies near the beginning of the respiration pathway in cells </li></ul><ul><li>The end product of respiration is ATP </li></ul><ul><li>If there is a lot of ATP in the cell this enzyme is inhibited </li></ul><ul><li>Respiration slows down and less ATP is produced </li></ul><ul><li>As ATP is used up the inhibition stops and the reaction speeds up again </li></ul>© 2008 Paul Billiet ODWS
    109. 123. The switch: Allosteric inhibition <ul><li>Allosteric means “other site” </li></ul>Active site Allosteric site © 2008 Paul Billiet ODWS E
    110. 124. Switching off <ul><li>These enzymes have two receptor sites </li></ul><ul><li>One site fits the substrate like other enzymes </li></ul><ul><li>The other site fits an inhibitor molecule </li></ul>Inhibitor fits into allosteric site Substrate cannot fit into the active site Inhibitor molecule © 2008 Paul Billiet ODWS
    111. 125. The allosteric site the enzyme “on-off” switch Active site Allosteric site empty Substrate fits into the active site The inhibitor molecule is absent Conformational change Inhibitor fits into allosteric site Substrate cannot fit into the active site Inhibitor molecule is present © 2008 Paul Billiet ODWS E E
    112. 126. A change in shape <ul><li>When the inhibitor is present it fits into its site and there is a conformational change in the enzyme molecule </li></ul><ul><li>The enzyme’s molecular shape changes </li></ul><ul><li>The active site of the substrate changes </li></ul><ul><li>The substrate cannot bind with the substrate </li></ul>© 2008 Paul Billiet ODWS
    113. 127. Negative feedback is achieved <ul><li>The reaction slows down </li></ul><ul><li>This is not competitive inhibition but it is reversible </li></ul><ul><li>When the inhibitor concentration diminishes the enzyme’s conformation changes back to its active form </li></ul>© 2008 Paul Billiet ODWS
    114. 128. Phosphofructokinase <ul><li>The respiration pathway accelerates and ATP (the final product) builds up in the cell </li></ul><ul><li>As the ATP increases, more and more ATP fits into the allosteric site of the phosphofructokinase molecules </li></ul><ul><li>The enzyme’s conformation changes again and stops accepting substrate molecules in the active site </li></ul><ul><li>Respiration slows down </li></ul>© 2008 Paul Billiet ODWS
    115. 132. Enzyme Inhibition (Mechanism) Competitive Non-competitive Uncompetitive E E Different site Compete for active site Inhibitor Substrate Cartoon Guide Equation and Description [ I ] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [ I ]. [ I ] binds to free [E] or [ES] complex; Increasing [S] can not overcome [ I ] inhibition. [ I ] binds to [ES] complex only, increasing [S] favors the inhibition by [ I ]. X Juang RH (2004) BCbasics E + S -> ES -> E + P + I ↓ E I ← ↑ E + S -> ES -> E + P + + I I ↓ ↓ E I + S ->E I S ← ↑ ↑ E + S -> ES -> E + P + I ↓ E I S ← ↑
    116. 133. Competitive Inhibition Succinate Glutarate Malonate Oxalate Succinate Dehydrogenase Substrate Competitive Inhibitor Product C-OO - C-H C-H C-OO - C-OO - H-C-H H-C-H C-OO - C-OO - H-C-H H-C-H H-C-H C-OO - C-OO - C-OO - C-OO - H-C-H C-OO -
    117. 134. Enzyme Inhibition (Plots) V max K m K m ’ [S], mM v o I I V max unchanged K m increased V max decreased K m unchanged Both V max & K m decreased I = K m ’ Juang RH (2004) BCbasics K m Competitive Non-competitive Uncompetitive Direct Plots Double Reciprocal V max [S], mM v o K m [S], mM V max I K m ’ V max ’ V max ’ 1/[S] 1/K m 1/ v o 1/ V max I Two parallel lines I Intersect at X axis 1/ v o 1/ V max 1/[S] 1/K m 1/[S] 1/K m 1/ V max 1/ v o Intersect at Y axis
    118. 135. The effect of enzyme inhibition <ul><li>Irreversible inhibitors : Combine with the functional groups of the amino acids in the active site, irreversibly. </li></ul><ul><li>Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase. </li></ul>© 2007 Paul Billiet ODWS
    119. 136. The effect of enzyme inhibition <ul><li>Reversible inhibitors : These can be washed out of the solution of enzyme by dialysis. </li></ul><ul><li>There are two categories. </li></ul>© 2007 Paul Billiet ODWS
    120. 137. The effect of enzyme inhibition <ul><li>Competitive : These compete with the substrate molecules for the active site. </li></ul><ul><li>The inhibitor’s action is proportional to its concentration. </li></ul><ul><li>Resembles the substrate’s structure closely. </li></ul>© 2007 Paul Billiet ODWS Enzyme inhibitor complex Reversible reaction E + I EI
    121. 138. CLINICAL APPLICATIONS OF COMPETITVE INHIBITORS METHANOL POISONING METHANOL Al.Dehy. ETHANOL ANTIBIOTIC PABA Dihydro pteroate Synthase SULFONAMIDE GOUT HYPOXANTHENE XANTHINE OXIDASE ALLOPURINOL Clinical App. TRUE SUB. ENZYME DRUG
    122. 139. The effect of enzyme inhibition <ul><li>Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. </li></ul><ul><li>Examples </li></ul><ul><li>Cyanide combines with the Iron in the enzymes cytochrome oxidase. </li></ul><ul><li>Heavy metals, Ag or Hg, combine with –SH groups. </li></ul><ul><li>These can be removed by using a chelating agent such as EDTA. </li></ul>© 2007 Paul Billiet ODWS
    123. 142. Applications of inhibitors <ul><li>Negative feedback : end point or end product inhibition </li></ul><ul><li>Poisons snake bite, plant alkaloids and nerve gases. </li></ul><ul><li>Medicine antibiotics, sulphonamides, sedatives and stimulants </li></ul>© 2007 Paul Billiet ODWS
    124. 143. Enzyme Inhibitors Are Extensively Used ● Sulfa drug (anti-inflammation) Pseudo substrate competitive inhibitor ● Protease inhibitor Plaques in brain contains protein inhibitor ● HIV protease is critical to life cycle of HIV HIV protease (homodimer): ↑ inhibitor is used to treat AIDS Symmetry Not symmetry -> Human aspartyl protease: (monodimer) Alzheimer's disease domain 1 Asp Asp domain 2 subunit 2 Asp subunit 1 Asp
    125. 144. DIAGNOSTIC SIGNIFICANCE <ul><li>ISOENZYMES: </li></ul><ul><li>1.CPK: MM,BB and MB.(Skeltol muscles,Brain and Myocardium) </li></ul><ul><li>2. LDH:LDH-1 (H4) Myocardium </li></ul><ul><li>LDH-2 (H3M) Erythrocytes. </li></ul><ul><li>LDH-3 (H2M2) Brain. </li></ul><ul><li>LDH-4 (HM3) Liver,Muscles. </li></ul><ul><li>LDH-5 (M4) Liver, Muscles. </li></ul>
    126. 145. <ul><li>Plasma enzymes are of two types: </li></ul><ul><li>A small group of enzymes secreted into the blood by certain cells e.g. the liver secretes zymogens (inactive form of enzymes) of blood coagulation. </li></ul><ul><li>FUNCTIONAL: Lipoprotein lipase,Pseudocholine estrase,blood coagulation. </li></ul><ul><li>NON FUNCTIONAL ENZYMES: </li></ul>
    127. 146. <ul><li>A large group of enzymes are released from cells during normal cell turnover. These enzymes function intracellularly (inside cells) and have no function in the blood. In healthy individuals, the blood levels of these enzymes are constant, as the rate of release from damaged cells into blood is equal to the rate of removal of enzymes from blood. </li></ul>
    128. 147. <ul><li>Elevated enzyme activity in blood indicates tissue damage (due to increased release of intracellular enzymes). </li></ul>
    129. 148. A. Plasma Enzymes as diagnostic tools <ul><li>Diseases that cause tissue damage result in increased release of intracellular enzymes into the plasma. </li></ul><ul><li>Determination of the level of these enzymes is used for diagnosis of heart, liver, skeletal muscle, etc. </li></ul><ul><li>The level of these enzymes in plasma correlates with the extent of tissue damage. </li></ul>
    130. 149. <ul><li>The presence of increased levels of some enzymes in plasma is diagnostic to damage of a particular tissue; e.g. The enzyme alanine aminotransferase (ALT) is abundant in the liver and the appearance of elevated levels of ALT in plasma indicates damage to the liver. </li></ul>
    131. 150. Intracellular Distribution of Diagnostic Enzymes ACP ALP GGT CK ALP AMS LPS AMS LD 1 AST CK LD 5 ALT AST Prostate Biliary Tract Muscle Bone Salivary Glands Pancreas Heart Liver
    132. 151. ISOENZYMES <ul><li>Isoenzymes or Isozymes are physically distinct form of same enzyme having same specificity, but are present in different tissues of same organism, in different cell compartment. </li></ul><ul><li>Useful for diagnosing diseases of different organs. </li></ul><ul><li>Homomultimer:All the units are same. </li></ul><ul><li>Heteromultimer:Sub units are different.These are produced by different genes. </li></ul>
    133. 152. IDENTIFICATION OF ISOZYMES <ul><li>1.Agar gel or PAGE.They have different mobility. </li></ul><ul><li>2.Heat stability. </li></ul><ul><li>3.Inhibitors.Isozymes may be sensative to different inhibitors.eg.tartrate labile. </li></ul><ul><li>4. Km value or substrate specificity. Eg.Glucokinase has high Km and Hexokinase has low Km for Glucose. </li></ul><ul><li>5.Co-Factors.Eg Mitochondrial isocitrate dehydrogenase is NAD dependent,Cytoplasmic NADP dependent. </li></ul><ul><li>6.Localisation:H4 heart,M4 Muscles. </li></ul><ul><li>7.Specific antibodies identify sp.Isozyme. </li></ul>
    134. 153. Isoenzymes <ul><li>Isoenzymes catalyze the same reaction in different tissues in the body. </li></ul><ul><li>Lactate dehydrogenase, which converts lactate to pyruvate, (LDH) consists of five isoenzymes. </li></ul>
    135. 154. Diagnostic Significance Enzymes <ul><li>The levels of diagnostic enzymes determine the amount of damage in tissues. </li></ul>
    136. 155. B. Isoenzymes and Heart Diseases <ul><li>Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction. </li></ul><ul><li>However, these enzymes do not have the same physical properties (as they differ in amino acid sequence). </li></ul><ul><li>Thus, they differ in electrophoretic mobility. </li></ul><ul><li>The plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial infarction. </li></ul>
    137. 156. DIAGNOSTIC IMPORTANCE <ul><li>Creatine phosphokinase </li></ul><ul><li>Alkaline phosphatase </li></ul><ul><li>CPK-BB,CPK-MM,CPK-MB </li></ul><ul><li>Alfa 1 ALP (liver) </li></ul><ul><li>Alfa 2 ALP Heat labile (Liver)& Heat stable (Placenta) </li></ul><ul><li>Prebeta ALP (Bones) </li></ul><ul><li>Gama ALP (Colon) </li></ul><ul><li>Regan ALP (Tumours) </li></ul>
    138. 157. DISORDERS DIAGNOSED BY ENZYMES <ul><li>1) Cardiac Disorders. </li></ul><ul><li>2) Hepatic Disorders. </li></ul><ul><li>3) Skeletal Muscle Disorders. </li></ul><ul><li>4) Bone Disorders. </li></ul><ul><li>5) Pancreatic Disorders. </li></ul><ul><li>6) Salivary gland diseae (Mumps) </li></ul><ul><li>7) Malignancies </li></ul>
    139. 158. CARDIAC MARKERS <ul><li>CPK (MB) </li></ul><ul><li>LDH (1) </li></ul><ul><li>CARDIAC TROPONIN (I)&(T) </li></ul><ul><li>BRAIN NATRIURETIC PEPTIDE </li></ul><ul><li>(Marker of Ventricular function) </li></ul><ul><li>AST </li></ul><ul><li>ALT </li></ul>
    140. 159. LIVER MARKERS <ul><li>ALT (Alanine amino transferase) </li></ul><ul><li>ALP (Alkaline phosphatase) </li></ul><ul><li>NTP (Nucleotide phosphatase) </li></ul><ul><li>GGT (Gama glutamyl Tranferase) </li></ul>
    141. 160. PROSTATE MAR <ul><li>PSA (prostate SP.ANTIGEN. </li></ul><ul><li>ACP (Acid Phosphatase) </li></ul>
    142. 161. MUSCLE MARKER <ul><li>CK (MM) </li></ul><ul><li>AST (Aspartate Amino Transferase) </li></ul><ul><li>ALD (Aldolase) </li></ul>
    143. 162. BONE MARKER <ul><li>ALP (Alkaline Phosphatase) </li></ul>
    144. 163. <ul><li>Cardiac Markers: </li></ul><ul><li>e.g. Acute Myocardial Infarction (AMI). </li></ul><ul><li>1) The myocardium becomes ischemic and </li></ul><ul><li>undergoes necrosis. </li></ul><ul><li>2) Cellular contents are released into the circulation. Blood levels of the following enzymes increase: </li></ul>CK LD 1 AST
    145. 164. 2. Hepatic Disorders <ul><li>Hepatocellular Disorders : </li></ul><ul><li>(1) Viral hepatitis: Hepatitis B & Hepatitis C. </li></ul><ul><li>(2) Toxic hepatitis: caused by chemicals & Toxins (e.g aflatoxin, Asp. flavus) </li></ul><ul><li>Increased levels of the following enzymes : </li></ul>LD 5 AST ALT
    146. 165. <ul><li>b) Biliary tract disorders : </li></ul><ul><li> The plasma levels of the following enzymes increase: </li></ul>GGT ALP
    147. 166. 3. Skeletal Muscle Disorders <ul><li>Muscle dystrophy. </li></ul><ul><li>Muscle trauma. </li></ul><ul><li>Muscle hypoxia. </li></ul><ul><li>Frequent I.M Injections. </li></ul><ul><li>The plasma levels of the following enzymes increase: </li></ul>AST CK
    148. 167. 4. Bone Disorders: <ul><li>1) Paget’s Bone Disease: caused by increased osteoclastic activity. </li></ul><ul><li>2) Rickets </li></ul><ul><li>3) Osteomalacia: The plasma levels of the following enzyme increase: </li></ul>ALP
    149. 168. 5. Acute Pancreatitis <ul><li>The plasma levels of the following enzymes increase: </li></ul>AMS Lipase
    150. 169. 6. Salivary Gland Inflammation: <ul><li>In Mumps: </li></ul><ul><li>The levels of  -Amylase (AMS) is significantly increased </li></ul>
    151. 170. 7. Malignancies <ul><li>Plasma (Acid phosphatase) ACP levels increase in: </li></ul><ul><li>Prostatic carcinoma. </li></ul><ul><li>Bone metastatic carcinoma </li></ul>
    152. 171. <ul><ul><li>b) Plasma levels of Alkaline phosphatase (ALP) increase in: </li></ul></ul><ul><ul><li>Pancreatic carcinoma. </li></ul></ul><ul><ul><li>Bile duct carcinoma. </li></ul></ul><ul><ul><li>Liver metastasis. </li></ul></ul>
    153. 172. <ul><ul><li>c) Plasma levels of Total Lactate dehydrogenase (LDH) increase in: </li></ul></ul><ul><ul><li>Leukemia </li></ul></ul><ul><ul><li>Lymphomas. </li></ul></ul><ul><ul><li>Liver metastasis. </li></ul></ul>
    154. 173. B. Isoenzymes and Heart Diseases <ul><li>Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction. </li></ul><ul><li>However, these enzymes do not have the same physical properties (as they differ in amino acid sequence). </li></ul><ul><li>Thus, they differ in electrophoretic mobility. </li></ul><ul><li>The plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial infarction. </li></ul>
    155. 174. <ul><li>Many isoenzymes contain different subunits in various combinations. </li></ul><ul><li>CK occurs in 3 isoenzymes, each is a dimer composed of 2 subunits (B & M): CK1 = BB, CK2 = MB and CK3 = MM, each CK isozyme shows a characteristic electrophoretic mobility. </li></ul>
    156. 175. <ul><li>Myocardial muscle is the only tissue that contains high level of CK2 (MB) isoenzyme. </li></ul><ul><li>Appearance of CK2(MB) in plasma is specific for heart infarction. </li></ul><ul><li>Following an acute myocardial infarction,CK2appears in plasma 4-8 hours following onset of chest pain (peak is reached after 24 hours). </li></ul>
    157. 176. Alkaline Phosphatase <ul><li>1.Alfa1-ALP Liver </li></ul><ul><li>2.Alfa2-ALP Liver (Heat Labile) </li></ul><ul><li>3.Pre Beta-ALP (BONES) </li></ul><ul><li>4.Gama ALP (Ulcerative Colitis) </li></ul><ul><li>5.Regan ALP (Bronchogenic cancer) </li></ul><ul><li>Sialic Acid Residues </li></ul>
    158. 177. ENZYMES IN OTHER BODY FLUIDS <ul><li>Adenosine deaminase in pleural fluid :Elevated in Tuberculosis not in Malignant effusion. </li></ul><ul><li>LDH; In CSF,Pleural fluid & Ascitic Fluid. </li></ul><ul><li>Elevated levels in Malignacy. </li></ul>
    159. 178. Enzymes as Therapeutic Agents <ul><li>Dissolving thrombus,Streptokinase,Urokinase. </li></ul><ul><li>Asparaginase used in some leukemias. </li></ul><ul><li>Deoxyribonuclease is adminstered via respiratory route to clear viscid secretions in pt. of cystic fibrosis. </li></ul><ul><li>Serratiopeptidase is used to minimise edema in acute inflamatory conditions. </li></ul><ul><li>Hyaluronidase for hypovolumia </li></ul><ul><li>Hemocoagulase used as hemostat. </li></ul><ul><li>Fungal Diastase &Pepsin used as digestive enz. </li></ul><ul><li>Ribozymes &Abzymes </li></ul><ul><li>Streptodornase; DNA applied locally. </li></ul><ul><li>Alpha-1-ant-trypsin; Emphysema. </li></ul>
    160. 179. ENZYMES USED FOR DIAGNOSIS <ul><li>Urease Urea. </li></ul><ul><li>Uricase Uric Acid. </li></ul><ul><li>Glucose Oxidase Glucose. </li></ul><ul><li>Peroxidase Cholesterol. </li></ul><ul><li>Hexokinase Glucose. </li></ul><ul><li>Lipase Triglycerides. </li></ul><ul><li>Alkaline phosphatase ELISA. </li></ul><ul><li>Restriction endonuclease RFLP </li></ul>
    161. 180. THANKS
    162. 181. Competitive Inhibition Succinate Glutarate Malonate Oxalate Succinate Dehydrogenase Substrate Competitive Inhibitor Product C-OO - C-H C-H C-OO - C-OO - H-C-H H-C-H C-OO - C-OO - H-C-H H-C-H H-C-H C-OO - C-OO - C-OO - C-OO - H-C-H C-OO -
    163. 182. Enzyme Active Site Is Deeper than Ab Binding Instead, active site on enzyme also recognizes substrate, but actually complementally fits the transition state and stabilized it. Ag binding site on Ab binds to Ag complementally, no further reaction occurs. X
    164. 183. cAMP Controls Activity of Protein Kinase A A A A A cAMP Active kinase CREB CREB Nucleus Activation Gene expression ON DNA Alberts et al (2002) Molecular Biology of the Cell (4e) p. 857, 858 R C R C R R A A A A C C Regulatory subunits Catalytic subunits C P
    165. 184. HIV protease vs Aspartyl protease Asymmetric monomer ↓ HIV protease (homodimer) HIV Protease inhibitor is used in treating AIDS Symmetric dimer ↑ Aspartyl protease (monomer) Juang RH (2004) BCbasics Asp subunit 2 subunit 1 Asp domain 1 domain 2 Asp Asp
    166. 185. Enzyme Inhibitors Are Extensively Used ● Sulfa drug (anti-inflammation) Pseudo substrate competitive inhibitor ● Protease inhibitor Plaques in brain contains protein inhibitor ● HIV protease is critical to life cycle of HIV HIV protease (homodimer): ↑ inhibitor is used to treat AIDS Symmetry Not symmetry -> Human aspartyl protease: (monodimer) Alzheimer's disease domain 1 Asp Asp domain 2 subunit 2 Asp subunit 1 Asp
    167. 186. Sulfa Drug Is Competitive Inhibitor Precursor Folic acid Tetrahydro- folic acid Sulfanilamide Sulfa drug (anti-inflammation) Para-aminobenzoic acid (PABA) Bacteria needs PABA for the biosynthesis of folic acid Sulfa drugs has similar structure with PABA, and inhibit bacteria growth. Domagk (1939) -COOH H 2 N- -SONH 2 H 2 N-
    168. 187. The Reception and Transduction of Signals -GDP +GTP Adenylate cyclase   Glycogen Synthase active Insulin kinase Glucagon A The third group: Ion-channel-linked Receptor Gilman, Rodbell (1994) Glycogen breakdown Juang RH (2007) BCbasics SH2 domain  G protein   GDP   + Signal  GDP  GTP  GTP + Signal Activation P Protein Phosphatase Glycogen Synthase P P P P P G-protein-linked Receptor Enzyme-linked Receptor Glycogen
    169. 188. Signal Transduction Network (Ras vs. P53) Cytosol Cell membrane Effector enzyme Signal protein E2F Transcription factor Target gene mRNA Inhibitor P53 Cell division ON Signal Receptor Nucleus Ribosome Transcription Transcription Apoptosis Cell function are controlled by protein interactions mRNA Regulator protein Juang RH (2007) BCbasics Ras
    170. 189. The Reception and Transduction of Signals -GDP +GTP Adenylate cyclase   Glycogen Synthase active Insulin kinase Glucagon A The third group: Ion-channel-linked Receptor Gilman, Rodbell (1994) Glycogen breakdown Juang RH (2007) BCbasics SH2 domain  G protein   GDP   + Signal  GDP  GTP  GTP + Signal Activation P Protein Phosphatase Glycogen Synthase P P P P P G-protein-linked Receptor Enzyme-linked Receptor Glycogen
    171. 190. A PKA active inactive Glucagon P P GP kinase GP kinase GP a GP b Glycogen synthase Glycogen synthase P Protein phosphatase-1 Protein phosphatase-1 Protein phosphatase inhibitor-1 Protein phosphatase inhibitor-1 Glycogen P Phosphatase
    172. 191. Classification of Proteases Metal Protease Serine Protease Cysteine Protease Aspartyl Protease   Family Example   Mechanism Specificity Inhibitor Juang RH (2004) BCbasics Carboxy- peptidase A Chymotrypsin Trypsin Papain Pepsin Renin H57 D102 S195-O - C25-S - H195 D215 D32 H 2 O Non- specific Non- specific Aromatic Basic Non- polar EDTA EGTA DFP TLCK TPCK PCMB Leupeptin Pepstatin E72 H69 Zn 2+ H196
    173. 192. Modification of Subtilisin and Its Activity Change No enzyme 1 Asn 155 -> Leu ● ● ● 10,000,000 ( Asn 155 stabilizes transition state ) His & Asp -> Ala ● ○ ○ 37,000 Ser , His & Asp -> Ala ○ ○ ○ 4,000 Subtilisin ● ● ● 10,000,000,000 Active Site Relative Modification Triad: Ser His Asp activity Ser -> Ala ○ ● ● 5,000 Asp -> Ala ● ● ○ 330,000
    174. 193. Serine Protease and AchE Chymotrypsin – Gly – Asp – Ser – Gly – Gly – Pro – Leu – Trypsin – Gly – Asp – Ser – Gly – Gly – Pro – Val – Elastase – Gly – Asp – Ser – Gly – Gly – Pro – Leu – Thrombin – Gly – Asp – Ser – Gly – Gly – Pro – Phe – Plasmin – Gly – Asp – Ser – Gly – Gly – Pro – Leu – Acetylcholinesterase – Gly – Glu – Ser – Ala – Gly – Gly – Ala – Chymotrypsin – Val – Thr – Ala – Ala – His – Cys – Gly – Trypsin – Val – Ser – Ala – Gly – His – Cys – Tyr – Elastase – Leu – Thr – Ala – Ala – His – Cys – Ile – Thrombin – Leu – Thr – Ala – Ala – His – Cys – Leu – Plasmin – Leu – Thr – Ala – Ala – His – Cys – Leu – Acetylcholinesterase – – – – – – – – – – – – – His – – – – – – – – Ser 195 Chymotrypsin – Thr – Ile – Asn – Asn – Asp – Ile – Thr – Trypsin – Tyr – Leu – Asn – Asn – Asp – Ile – Met – Elastase – Ser – Lys – Gly – Asn – Asp – Ile – Ala – Thrombin – Asn – Leu – Asp – Arg – Asp – Ile – Ala – Plasmin – Phe – Thr – Arg – Lys – Asp – Ile – Ala – Acetylcholinesterase – – – – – – – – – – – – – – Asp – – – – – – – His 57 Asp 102 Adapted from Dressler & Potter (1991) Discovering Enzymes, p.244
    175. 194. H AchE Has Similar Catalytic Mechanism H - O - H H 2 O Adapted from Dressler & Potter (1991) Discovering Enzymes, p.243 ↓ Deacylation Acylation↑ AchE O - C H O CH 3 CH 3 – C – O–CH 2 –CH 2 – N –CH 3 CH 3 + AchE O C H O CH 3 – C CH 3 H O–CH 2 –CH 2 – N –CH 3 CH 3 + AchE O - C H H O CH 3 – C – OH AchE O - C H O CH 3 – C CH 3 O–CH 2 –CH 2 – N –CH 3 H CH 3 +
    176. 195. Different Enzymes Might Adopt Same Mechanism Hi, Everybody! ← Useful ↙ Amusing Juang RH (2004) BCbasics O - C Sesame Triad
    177. 196. Convergent and Divergent Trypsin Chymotrypsin Elastase Thrombin Plasmin Acetylcholin esterase Thyroglobulin Ester bond Peptide bond hydrolyze acetylcholine Serine Protease Juang RH (2004) BCbasics Divergent evolution Asp-- His-- Ser Asp--His--Ser Convergent evolution C N C C H O C C C O O Evolution Molecular
    178. 197. Activity Regulation of Glycogen Phosphorylase Covalent modification P P GP kinase GP phosphatase 1 Non-covalent A A A AMP ATP Glc-6-P Glucose Caffeine Glucose Caffeine spontaneously R T R T Garrett & Grisham (1999) Biochemistry (2e) p.679 P A P A P P A A P A P A P P P A P A
    179. 198. CCC Allosteric Enzyme ATCase + Active relaxed form Inactive tense form ATCase R R R R R R CCC Catalytic subunits Catalytic subunits Regulatory subunits Aspartate Carbamoyl phosphate Carbamoyl aspartate Juang RH (2004) BCbasics Quaternary structure COO - CH 2 HN-C-COO - H H - - - - O H 2 N-C-O-PO 3 2- = O H 2 N-C- = COO - CH 2 N-C-COO - H H - - - - ATP CTP Nucleic acid metabolism Feedback inhibition CTP CTP CTP CTP CTP CTP
    180. 199. Regulation of Enzyme Activity P R R + proteolysis phosphorylation cAMP or calmodulin or regulator effector (+) Juang RH (2004) BCbasics Regulatory subunit or inhibitor P (-) Inhibitor Proteolysis Phosophorylation Signal transduction Feedback regulation

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