生物化學(xué)本科課件英文版講授chapter

1、Chapter 9 Catalytic StrategiesWhat are the sources of the catalytic power and selectivity of enzymes? 酶的高效專一源于什么?the catalytic strategies used by several well-understood enzymes: lysozyme溶菌酶, ribonuclease A 核糖核酸酶A, carboxypeptidase羧肽酶, and chymotrypsin胰凝乳蛋白酶. The three-dimensional structures of thes

2、e enzymes are known in atomic detail, as is their mode of binding of substrate analogs and inhibitors. 已知上述酶原子水平的三維結(jié)構(gòu)及其與底物類似物和抑制劑的結(jié)合模式.The reactions catalyzed are relatively simplenamely, the hydrolysis of glycosidic糖苷鍵, phosphodiester磷酸二酯鍵, and peptide bonds肽鍵. Each of these enzymes digests a macro

3、molecular substrate and exemplifies an important category of hydrolytic reactions都水解大分子底物. The actions of these enzymes illustrate many important principles of catalysis. enzymes facilitate the formation of transition states.Design potent and specific enzyme inhibitors A strategy for inhibiting the

4、HIV-1 protease蛋白酶, which is essential for replication of the AIDS virus, is presented as an example. a brief account of the discovery of catalytic RNA and its biological significance.FLEMINGS DISCOVERY OF LYSOZYMEAlexander Fleming found that the bacteria near his mucus鼻粘膜 had been dissolved away.the

5、 antibacterial substance was an enzyme. He also discovered a small round bacterium (Micrococcus 微球菌微球菌lysodeikticus) particularly susceptible to lysozymeTears are a rich source of lysozymeLysozyme溶菌酶 was not effective against the most harmful bacteria. But seven years later, he did discover a highly

6、 effective antibiotic, penicillina striking illustration of Pasteurs comment that chance favors the prepared mind.LYSOZYME CLEAVES BACTERIAL CELL WALLSLysozyme dissolves certain bacteria by cleaving the polysaccharide component of their cell walls. The function of the cell wall in bacteria is to con

7、fer mechanical support. A bacteria cell devoid of a cell wall usually bursts because of the high internal osmotic pressure. Penicillin blocks cell wall synthesis by inactivating a transpeptidase轉(zhuǎn)肽酶 that catalyzes the formation of essential cross-links between peptide units.溶菌酶切斷細(xì)菌細(xì)胞壁多糖鏈?zhǔn)鼓承┘?xì)菌裂解,青霉素則使

8、(催化肽鏈交聯(lián)的)轉(zhuǎn)肽酶失活而阻斷細(xì)菌細(xì)胞壁的合成.The cell wall polysaccharide is made up of two kinds of sugars: N-acetylglucosamine (NAG)乙酰葡糖胺 and N-acetylmuramate (NAM)乙酰胞壁酸(乙酸,葡糖胺,乳酸) NAM and NAG are derivatives of glucosamine in which the amino group is acetylated. In bacterial cell walls, NAM and NAG are joined by gl

9、ycosidic linkages. The oxygen atom in a glycosidic bond can be located either above or below the plane of the sugar ring. ( configuration, below the plane; configuration, above). All glycosidic bonds of the cell wall polysaccharide have a configuration. NAM and NAG alternate in sequence. Thus, the c

10、ell wall polysaccharide is an alternating polymer of NAM and NAG residues joined by (14) glycosidic linkages.Polysaccharide chains have directionality. Different polysaccharide chains are cross-linked by short peptides that are attached to NAM residues. Lysozyme, a glycosidase糖苷酶糖苷酶, hydrolyzes the

11、glycosidic bond between C-1 of NAM and C-4 of NAG . 它能催化水解它能催化水解HAM的的C1與與NAG的的C4之間的糖苷鍵之間的糖苷鍵,但不能水解但不能水解HAG的的C1與與NAM的的C4之間的之間的(1-4) 糖苷鍵糖苷鍵.Chitin殼多糖, a polysaccharide found in the shell of crustaceans甲殼類, is also a substrate for lysozyme. Chitin consists only of NAG residues joined by (14) glycosidic

12、 links.LYSOZYME IS A COMPACT PROTEIN WITH A COMPLEX FOLDLysozyme is a relatively small enzyme (14.6 kd). This highly stable protein (single chain of 129 residues )is cross-linked by four disulfide bridges. Lysozyme is a compact molecule, roughly ellipsoidal in shape橢圓體, with dimensions 453030. The f

13、olding of this protein is complex. There is much less helix than in myoglobin and hemoglobin. In a number of regions, the polypeptide chain is in an extended sheet conformation. 折疊片構(gòu)象The interior of lysozyme, like that of myoglobin and hemoglobin, is almost entirely nonpolar. 內(nèi)部幾乎完全非極性Hydrophobic in

14、teractions play an important role in the folding of lysozyme, as they do for most proteins.疏水作用FINDING THE ACTIVE SITE IN LYSOZYMELysozyme does not contain a prosthetic group and thus lacks a built-in marker 自設(shè)標(biāo)記at its active site.The essential information came from an x-ray crystallographic study o

15、f the interaction of lysozyme with inhibitors. When the three-dimensional structure of a protein is known, the mode of binding of small molecules can often be determined quite readily by x-ray crystallographic methods. much information can be derived from a study of a complex of an enzyme with an un

16、reactive (or very slowly reactive) analog of its substrate. For lysozyme, this was achieved with the trimer of N-acetylglucosamine (tri-NAG, or NAG3). 三聚體NAGn(n5) are hydrolyzed very slowly or not at all. However, they do bind to the active site of the enzyme. Indeed, tri-NAG is a potent competitive

17、 inhibitor of lysozyme. 因NAG3幾乎不(被溶菌酶)水解且確實(shí)與酶的活性部位結(jié)合,而溶菌酶因不含輔基(即不存在自設(shè)標(biāo)記)難以對(duì)活性部位定位,所以NAG3可作為研究溶菌酶活性部位的理想材料.MODE OF BINDING OF NAG3, A COMPETITIVE INHIBITORThe x-ray study of the tri-NAG complex with lysozyme showed the location of the active site活性部位位置, revealed the interactions responsible for the s

18、pecific binding of substrate特定結(jié)合底物的作用方式, and led to the proposal of a detailed enzymatic mechanism酶促機(jī)制的細(xì)節(jié). Tri-NAG binds to lysozyme in a cleft at the surface of the enzyme. NAG3 結(jié)合在酶表面的裂隙中Tri-NAG is bound to lysozyme by hydrogen bonds and van der Waals interactions. Electrostatic interactions canno

19、t occur because tri-NAG lacks ionic groups.氫鍵和范氏力,而非靜電力維系其結(jié)合. The hydrogen bonds between tri-NAG and lysozyme (between Asp101 and A and B; 4 bonds with C)Van der Waals contacts: sugar residue B fits closely to the indole ring of tryptophan 57.色氨酸的吲哚環(huán)FROM STRUCTURE TO THE ENZYME MECHANISM OF LYSOZYME

20、1.How would a substrate bind? the structure of an enzyme complex with a competitive inhibitor provides clues to how a substrate binds. The finding that tri-NAG fills only half the cleft in lysozyme was a very suggestive starting point. 三糖NAG只占據(jù)酶裂隙的一半It seemed likely that additional sugar residues, w

21、hich would fill the rest of the cleft, were required for formation of a reactive ES complex. There was space for three additional sugar residues. This was encouraging, because it was known that hexa-NAG is rapidly hydrolyzed by the enzyme. 六糖才能填滿酶裂隙,形成ES復(fù)合物,被酶迅速水解Three additional sugar residues, nam

22、ed D, E, and F, were fitted into the cleft by careful model building. Residues E and F went in nicely, making a number of good hydrogen bonds and van der Waals contacts. However, residue D would not fit unless it was distorted.扭曲 六糖殘基為合適底物,其中C必須是NAG,D(NAG)必須經(jīng)扭曲為半椅式構(gòu)象2.Which bond is cleaved? The rate

23、 of hydrolysis of oligomers of NAG increases strikingly as the number of residues is increased from four to fivethat is, from NAG4 to NAG5. The cleavage rate increases further when a sixth residue is added (NAG6), but it stays the same as the number of residues is increased to eight. This finding is

24、 consistent with the crystallographic晶體學(xué) result showing that the active-site cleft would be fully occupied by six sugar residues.活性部位裂隙被六糖殘基完全占據(jù) the D-E bond was the only remaining candidate for the cleavage site.3.Which groups on the enzyme participate in catalysis? it is the D-E bond that is split

25、 on which side of the glycosidic oxygen atom is the bond cleaved? the bond split is the one between C-1 of residue D and the oxygen of the glycosidic linkage to residue E. (DC1-O-EC4) 切斷發(fā)生在切斷發(fā)生在D殘基殘基C1和糖苷鍵氧原子之間和糖苷鍵氧原子之間A search was then made for possible catalytic groups close to the glycosidic bond

26、 that is cleaved. A catalytic group is one that directly participates in making or breaking covalent bonds.The donation or abstraction of a hydrogen ion is a critical step in most enzymatic reactions.Hence, the most likely candidates are groups that serve as proton donors or acceptors.可能參與催化的殘基是(1)最

27、靠近被切割鍵,(2)并作為質(zhì)子供體或受體.The only plausible catalytic residues near the glycosidic bond that is cleaved by lysozyme are Asp 52 and Glu35. A CARBONIUM ION正碳離子正碳離子 INTERMEDIATE IS CRITICAL FOR CATALYSISa detailed catalytic mechanism for lysozyme based on the preceding structural data. The essential steps

28、areThe COOH of Glu 35 donates an H+ to the glycosidic oxygen atom between D and E. Glu35(向糖苷鍵氧向糖苷鍵氧)提供質(zhì)子并切斷提供質(zhì)子并切斷DE之間的鍵之間的鍵This creates a positive charge on C-1 of the D. This transient species is called a carbonium ion (or oxocarbonium ion) D環(huán)環(huán)C1帶正電荷帶正電荷,稱為正碳離子稱為正碳離子The dimer of E and F diffused a

29、way from the enzyme.The carbonium ion intermediate then reacts with OH (or H2O) from the solvent. Glu35 becomes reprotonated, and ABCD, diffuses away from the enzyme. Lysozyme is then ready for another round of catalysis. EF二聚體離開酶二聚體離開酶,正碳離子正碳離子與溶劑中與溶劑中OH作用作用, Glu35再質(zhì)子化再質(zhì)子化,ABCD四聚體離開酶四聚體離開酶The criti

30、cal elements of this proposed catalytic scheme are General acid catalysis.總酸催化(一般,廣義酸催化) The term general acid indicates that the source of the donor is a donor group rather than free H +. (specific acid catalysis)Promotion of the formation of the carbonium ion intermediate. The enzymatic reaction i

31、s markedly facilitated by two different factors that stabilize the carbonium ion intermediate: a. The electrostatic factor is the presence of a negatively charged group of Asp 52. b. The geometrical factor is the distortion of ring D. Hexa-NAG fits best into the active-site cleft if sugar residue D

32、is distorted out of its customary chair conformation into a half-chair form. This distortion enhances catalysis because the half-chair geometry markedly promotes the formation of the carbonium ion. 椅式半椅式THE PROPOSED MECHANISM IS SUPPORTED BY MANY LINES OF EXPERIMENTAL EVIDENCEThe mode of binding of

33、substrate and the mechanism of catalysis proposed on the basis of the crystallographic晶體學(xué) studies is supported by a variety of chemical experiments:Cleavage pattern. NAG6 is split into tetra- and di-NAG. 64+2Transition state analogs the distortion of the D ring of a normal substrate could accelerate

34、 catalysis several thousandfold. D環(huán)變形環(huán)變形Dependence of the catalytic rate on pH. The rate of hydrolysis of chitin (poly-NAG) is most rapid at pH 5. (Typical pK of the carboxyl group of Glu and Asp is 4.4)溶菌酶只有這樣才具活性: Glu35非離子化, Asp52離子化; Selective chemical modification. Esterification酯化 of aspartate

35、52 leads to a total loss of catalytic activity. Asp52修飾導(dǎo)致酶活性完全喪失X-ray evidence. NAM-NAG-NAM occupies the B-C-D sites rather than the A-B-C sites because NAM cannot fit into site C. NAM in site D is distorted from the chair to the sofa conformation .Thus, the bound sugar is forced into a conformation

36、 resembling that of the transition state. C位必須是NAG, D位NAM必須將構(gòu)象改變成過渡態(tài)半椅式. D環(huán)由正常的椅式變形為能量較高的半椅式構(gòu)象,從而糖苷鍵的穩(wěn)定性降低,鍵容易在這里被切斷.A CYCLIC PHOSPHATE INTERMEDIATE環(huán)磷酸中間物環(huán)磷酸中間物 IS FORMED IN THE HYDROLYSIS OF RNA BY RIBONUCLEASE Aribonuclease A, a digestive enzyme secreted by the pancreas 胰腺It has been intensively s

37、tudied because it is small (124 residues, 13.7 kd), plentiful, and stable. The enzyme can be cleaved by subtilisin枯草桿菌蛋白酶 at a single peptide bond to yield ribonuclease S, a catalytically active complex consisting of an S-peptide moiety (residues 1-20) and an S-protein moiety (residues 21-124) bound

38、 together by multiple noncovalent interactions.核糖核酸酶核糖核酸酶A經(jīng)切割后形成核糖核酸酶經(jīng)切割后形成核糖核酸酶S,核糖核酸酶核糖核酸酶S由由S蛋白蛋白(21124)和和S肽肽(120)組成組成.核糖核酸酶核糖核酸酶A和酶和酶S具有幾乎相同具有幾乎相同的酶活性的酶活性.Neither the S-peptide nor the S-protein alone is active. 單獨(dú)S蛋白和S肽都不具活性The catalytic properties of ribonuclease A and S are nearly the same, a

39、nd so we shall refer to them as ribonuclease.Ribonuclease catalyzes the hydrolysis of phosphodiester bonds in RNA chains. The bonds cleaved are ones between phosphorus and 5-oxygenatoms. One of the new termini formed has a free 5OH group, and the other has a free 3-phosphate group. Ribonuclease-cata

40、lyzed hydrolysis, like base-catalyzed hydrolysis in the absence of enzyme, proceeds through a 2,3-cyclic phosphate intermediate.核糖核酸酶催化和堿催化核糖核酸酶催化和堿催化RNA水解都經(jīng)過水解都經(jīng)過2,3-環(huán)磷酸中環(huán)磷酸中間物間物 The nucleotide on the 3 side of the scissile bond must be a pyrimidine, because a puring ring is too large to be accommo

41、dated in the active site without distorting it. 被切斷鍵的3 側(cè)必須是嘧啶因活性部位容納不了太大的嘌呤A uracil or cytosine ring binds to the active site by multiple, precisely directed hydrogen bonds. In contrast, ribonuclease is indifferent to the nature of all other bases in its RNA substrate because binding is primarily el

42、ectrostatic. A series of nine positively charged lysine and arginine side chains form salt bridges with the negatively charged phosphate backbone of RNA. Single-stranded DNA also binds to ribonuclease, but it is not hydrolyzed because DNA lacks a 2-hydroxyl group and hence cannot form a 2,3-cyclic i

43、ntermediate.單鏈DNA也可與核糖核酸酶結(jié)合,但不被水解,因它無(wú)2-羥基從而無(wú)法形成2,3-環(huán)磷酸中間物.A plot of the catalytic rate versus pH is bell-shaped, like that of lysozyme. Because the pH optimum is about 7, it was proposed that two histidine residues participate in catalysis, one in the basic form and the other in the acidic form. 兩個(gè)組

44、氨酸殘基參與催化(His12, His119)分別作為質(zhì)子供體和受體(pK of imidazole group咪唑基 of His is 6.5)Chemical modification studies supported this notion. Reaction of ribonuclease with iodoacetate碘乙酸, an alkylating reagent烷化劑, led to the carboxymethylation羧甲基化 of the imidazole ring of histidine 119 or histidine 12 but not both

45、 in the same enzyme molecule. 上述兩殘基之一被修飾都導(dǎo)致酶失活Modification of either histidine side chain inactivated the enzyme. Substrates or competitive inhibitors protected ribonuclease from modification and inactivation by iodoacetate. histidines 12 and 119 are near each other in the active site and act in con

46、cert as proton donors and acceptors in catalyzing the formation of the cyclic intermediate and its subsequent hydrolysis.PHOSPHORUS IS PENTACOVALENT五價(jià)五價(jià) IN THE TRANSITION STATE FOR RNA HYDROLYSISthe phosphonate膦酸鹽 analog of UpC (very similar to UpC), in which a methylene group (-CH2-) replaces the o

47、xygen atom in the scissile bond cannot be hydrolyzed by ribonuclease. The high-resolution structure of this enzyme-analog complex has been very valuable in formulating a detailed catalytic mechanism.用底物類似物(它不被核糖核酸酶水解)來(lái)研究催化機(jī)制的細(xì)節(jié)Three residues play a critical role in catalysis: histidine 12, histidine

48、 119 and lysine 41. In each stage, one apex is occupied by the attacking nucleophile親核基團(tuán)親核基團(tuán) and the other by the leaving group. CARBOXYPEPTIDASE A IS A ZINC-CONTAINING PROTEOLYTIC ENZYMEcarboxypeptidase A羧肽酶A, a digestive enzyme that hydrolyzes the carboxyl-terminal peptide bond in polypeptide chai

49、ns. Hydrolysis occurs most readily if the carboxyl-terminal residue has an aromatic or a bulky aliphatic side chain具有芳香族或較大脂族側(cè)鏈的羧基端殘基最容易被羧肽酶A水解This enzyme is a single polypeptide chain of 307 amino acid residues, has a compact shape that approximates an ellipsoid橢園體. The enzyme contains regions of h

50、elix (38%) and of pleated sheet (17%). A tightly bound zinc ion is essential for enzymatic activity. This zinc ion is located in a groove near the surface of the molecule, where it is coordinated配位 to two histidine side chains, a glutamate side chain, and a water molecule. 與兩個(gè)His,一個(gè)Glu,一個(gè)水配位A large

51、pocket near the zinc ion accommodates the side chain of the terminal residue of the peptide substrate.Two facets of the catalytic mechanism of carboxypeptidase A are particularly noteworthy:Induced fit. The binding of substrate is accompanied by many changes in the structure of the enzyme.Activation

52、 of water. The zinc ion at the active site markedly increases the reactivity of the bound water molecule.BINDING OF SUBSTRATE INDUCES LARGE STRUCTURAL CHANGES AT THE ACTIVE STITE OF CARBOXYPEPTIDASE A THE ZINC ION AT THE ACTIVE SITE ACTIVATES A WATER MOLECULEProteins and peptides are stable at neutr

53、al pH in the absence of a protease because water does not readily attack peptide bonds. A key catalytic task of carboxypeptidase A is to activate water. This is accomplished by the bound zinc ion with assistance from the adjacent carboxylate of glutamate 270. In fact, the zinc-bound H2O behaves much

54、 like an OH ion. 鋅離子(在臨近的谷氨酸協(xié)助下與水結(jié)合)激活水分子The first step in catalysis is the attack of the activated water molecule on the carbonyl group on the scissile peptide bond. 激活的水分子進(jìn)攻肽鍵(中的羰基)A negatively charged tetrahedral intermediate is formed. The next step is the transfer of a proton from the COOH grou

55、p of glutamate 270 to the peptide NH group. The peptide bond is concomitantly cleaved, and the reaction products diffuse away from the active site. The need for substrate-induced structural changes in the active site of carboxypeptidase A can now be appreciated. The bound substrate is surrounded on

56、all sides by catalytic groups of the enzyme. 鋅離子(在Glu270協(xié)助下)激活水分子被激活的水分子進(jìn)攻肽鍵(中的羰基) 帶負(fù)電荷的四面體中間物形成 Glu270向肽鍵(中的胺基)提供質(zhì)子 肽鍵被切斷,產(chǎn)物離開活性中心This arrangement promotes catalysis in three ways : (1) activation of H2O by Zn2+, (2) proton abstraction and donation by glutamate 270, and (3) electrostatic stabilizat

57、ion by arginine 127. It is evident that a substrate could not enter such an array of catalytic groups (nor could a product leave) unless the enzyme were flexible. A flexible protein provides a much larger repertoire of potentially catalytic conformations than does a rigid one.CHYMOTRYPSIN糜蛋白酶糜蛋白酶(胰凝

58、乳蛋胰凝乳蛋白酶白酶) IS A SERINE PROTEASEChymotrypsin, like carboxypeptidase A, is a mammalian digestive enzyme. However, its specificity and catalytic mechanism are quite different. Its biological role is to catalyze the hydrolysis of proteins in the small intestine. It is selective for peptide bonds on the

59、 carboxyl side of the aromatic side chains of tyrosine, tryptophan, and phenylalanine, and of large hydrophobic residues such as methionine. (Ala-phe-Asn-Ser-Met-Glu)Chymotrypsin has further significance as a member of an important family of proteins, the serine proteases. 絲氨酸蛋白酶家族成員Trypsin胰蛋白酶, ano

60、ther digestive enzyme, and thrombin凝血酶, a clotting enzyme, are other members of this pervasive clan. Chymotrypsin, a 25-kd enzyme, consists of three polypeptide chains connected by two interchain disulfide bonds.三條多肽鏈由兩個(gè)鏈間二硫鍵連接而成 it is synthesized as a singlechain inactive precursor called chymotryp