(62)--医学细胞生物学Chapter11HowProteinsWork.pdf

《(62)--医学细胞生物学Chapter11HowProteinsWork.pdf》由会员分享，可在线阅读，更多相关《(62)--医学细胞生物学Chapter11HowProteinsWork.pdf（20页珍藏版）》请在淘文阁 - 分享文档赚钱的网站上搜索。

1、P1:GIGWY001-11WY001-Bolsover-v2.clsSeptember 15,200321:3111HOW PROTEINS WORKThe three-dimensional structures of proteins generate binding sites for other molecules.This reversible binding is central to most of the biological roles of proteins,whether theprotein is a gap junction channel that binds a

2、 similar channel on another cell(page 55)ora transcription factor that binds to DNA.One special class of proteins,enzymes,have sitesthat not only bind another molecule but then catalyze a chemical reaction involving thatmolecule.How Proteins Bind Other MoleculesProteins can bind other protein molecu

3、les,DNA or RNA,polysaccharides,lipids,and avery large number of other small molecules and inorganic ions and can even bind dissolvedgases such as oxygen,nitrogen,and nitric oxide.Binding sites are usually very specific fora particular ligand,although the degree of specificity can vary widely.Usually

4、 the bindingis reversible so that there is an equilibrium between the free and bound ligand.A binding site is usually a cleft or pocket in the surface of the protein molecule,whichis made up of amino acid side chains appropriately positioned to make specific interactionswith the ligand.All of the fo

5、rces that stabilize tertiary structures of proteins are also usedin ligandprotein interaction:Hydrogen bonds,electrostatic interactions,the hydrophobiceffect,and van der Waals forces all have their roles.Even covalent bonds may be formedin a few casessome enzymes form a transient covalent bond with

6、the substrate as part ofthe mechanism used to effect the reaction.Cell Biology:A Short Course,Second Edition,by Stephen R.Bolsover,Jeremy S.Hyams,Elizabeth A.Shephard,Hugh A.White,Claudia G.WiedemannISBN 0-471-26393-1 CopyrightC?2004 by John Wiley&Sons,Inc.237P1:GIGWY001-11WY001-Bolsover-v2.clsSepte

7、mber 15,200321:31238HOW PROTEINS WORKglucoseglucoseFigure 11.1.The glucose carrier switches easily between two shapes.Dynamic Protein StructuresItiseasytogettheimpressionthatproteinstructuresarefixedandimmobile.Infactproteinsare always flexing and changing their structure slightly around their lowes

8、t energy state.A good term for this is“breathing.”Many proteins have two low-energy states in whichthey spend most of their time,like a sleeper who,though twisting and turning throughoutthe night,nevertheless spends most time lying on their back or side.An example is theglucose carrier(Fig.11.1).Thi

9、s is a transmembrane protein that forms a tube through themembrane.It is stable in one of two configurations.In one the tube is open to the cytosol;in the other the tube is open to the extracellular medium.By switching between the twostates,the glucose carrier carries glucose into and out of the cel

10、l.Allosteric EffectsThe glucose carrier is able to bind a ligandthe glucose moleculein either of its low-energy conformations.In contrast,the lac repressor(page 113)can only bind its ligand,theoperatorregionofthelacoperon,inoneconformation.Onitsowntheproteinpredominantlyadopts this conformation so t

11、ranscription is prevented as it binds to the DNA.When thelac repressor binds allolactose(a signal that lactose is abundant),it is locked into a second,inactiveformthatcannotbindtotheDNA(Fig.6.8onpage113).Transcriptionisnolongerrepressed,although the cAMPCAP complex is additionally required if transc

12、ription is toproceed at a high rate.This type of interaction,in which the binding of a ligand at oneplace affects the ability of the protein to bind another ligand at another location,is calledallostericandisusuallyapropertyofproteinswithaquaternarystructure(i.e.,withmultiplesubunits).Hemoglobin(Fig

13、.9.20 on page 205)is an example of a protein where allosteric effectsplay an important role.Each heme prosthetic group,one on each of the four subunits,canbindanoxygenmolecule.Wecangetanideaofwhatonesubunitonitsowncandobylookingat myoglobin(Fig.11.2a),a related molecule that moves oxygen within the

14、cytoplasm.Myoglobin has just one polypeptide chain and one heme.The green line in Figure 11.2bshows the oxygen-binding curve for myoglobin.Starting from zero oxygen,the first smallincrease in oxygen concentration produces a large amount of binding to myoglobin;theP1:GIGWY001-11WY001-Bolsover-v2.clsS

15、eptember 15,200321:31O2 pressure in pascal20000020406080100(a)(b)%of bindingsites occupied40006000hemoglobinmyoglobinFigure 11.2.(a)The monomeric oxygen-carrying protein myoglobin.(Illustration:Irving Geis.Rights owned by Howard Hughes Medical Institute.Reproduction by permission only.)(b)Oxy-gen bi

16、nding of myoglobin(in green)and hemoglobin(in black)as oxygen pressure increases.239P1:GIGWY001-11WY001-Bolsover-v2.clsSeptember 15,200321:31240HOW PROTEINS WORKnext increase in oxygen produces a slightly smaller amount of binding,and so on,untilmyoglobin is fully loaded with oxygen.A curve of this

17、shape is known as hyperbolic.Theblack line in Figure 11.2b shows the oxygen-binding curve for hemoglobin.Starting fromzero oxygen,the first small increase in oxygen concentration produces hardly any bindingto hemoglobin.The next increase in oxygen produces much more binding so the curve getssteeper

18、before leveling off again as the hemoglobin becomes fully loaded.This behavior iscalled cooperative.The explanation for this behavior is that the hemoglobin subunits canexist in one of two states,only one of which has a high affinity for oxygen.The way thatthe four subunits fit together means that t

19、hey all must be in one form or the other.Whenoxygen concentration is low,most of the hemoglobin molecules have their subunits in thelow-affinity form.As oxygen increases,it begins to bind to the hemoglobinlittle at firstas most of the hemoglobin is in the low-affinity form and only a little in the h

20、igh-affinityform.As oxygen binds,more molecules switch to the high-affinity form as the low-andhigh-affinity forms are in equilibrium.Eventually virtually all of the molecules have madethe switch to the high-affinity form.This produces the curve shown in Figure 11.2b.Thiscooperative oxygen binding m

21、akes hemoglobin an effective transporter as it will load upwith oxygen in the lungs but will release it readily in the capillaries of the tissues wherethe oxygen concentration is low.Myoglobin would release little of its bound oxygen at theoxygen concentrations typical of respiring tissues.Some enzy

22、mes show cooperative behavior caused by an allosteric effect that causesbinding of one substrate molecule to make it easier for the other substrate to bind.Thedegree of cooperativity can be altered by the binding of other molecules(called effectors)that act to switch the enzyme on or off.Chemical Ch

23、anges That Shift the Preferred Shape of a ProteinProteinscanchangeconformationasaresultofenvironmentalchanges,bybindingaligandor by having a particular group attached covalently to them.Anything that changes thepattern of electrostatic interactions within a protein will alter the relative energy of

24、itsstates.If,for example,a protein contains histidine residues,merely changing the pH will dothis.In solutions with pH greater than 7 most of the histidine residues in a protein will behistidineresiduespH8pH6+Figure11.3.A pH change that alters the charge on histidine will alter the balance of forces

25、 withina protein.At pH 6,the structure on the right will predominate.P1:GIGWY001-11WY001-Bolsover-v2.clsSeptember 15,200321:31ENZYMES ARE PROTEIN CATALYSTS241aspartateresidueATP heldnon-covalentlyin creviceResting statephosphorylatedaspartateresiduePhosphorylatedstateADPchargesrepel phosphate of ATP

26、transferred to aspartateresidue+CCOOOOCCOOOOOPOOPOOPOOOOOOPOOPOOOOOPOOFigure 11.4.Phosphorylation changes the charge pattern,and hence the balance of forces withinthe calcium ATPase,forcing a change of shape.uncharged(page 187).In solutions with a pH less than 7,most of the histidine residues willbe

27、arapositivecharge.Aproteinconformationthathadtwohistidineresiduesclosetogether(Fig.11.3)would therefore be stable in alkaline conditions,but in acid conditions the tworesidues would each bear a positive charge and repel,destabilizing the conformation.Anothermechanismthatisusedtochangetheconformation

28、ofproteinsistheadditionofanegativelychargedphosphategroupbyaclassofenzymescalledproteinkinases.Proteinscan be phosphorylated on serine,threonine,tyrosine,aspartate,glutamate,or histidine.Thecalcium ATPase(Fig.11.4)is a transmembrane protein that forms a tube that can be opento the cytosol or to the

29、extracellular medium.In its resting state the tube is open to thecytosol,while ATP is held by noncovalent interactions in a crevice in a cytosolic domain.Phosphorylation transfers the phosphate of ATP to a nearby aspartate residue.Repulsionbetween the phosphoaspartate and the remaining ADP means tha

30、t the lowest energy stateof the protein is now one in which the tube is open to the extracellular medium.Thismechanism is used to force calcium ions out of the cytoplasm into the extracellular fluid.The calcium ATPase is described in more detail on page 318.The movements produced in a protein by pho

31、sphorylation are small,on the order ofnanometers or less.When these are repeated many times and amplified by lever systems,they can produce movements of micrometers or even meters.The beating of a flagellum(page 386)or the kicking of a leg are both produced by phosphorylation-induced proteinshape ch

32、anges.ENZYMES ARE PROTEIN CATALYSTSLife depends on complex networks of chemical reactions.These are mediated by enzymes.Enzymes are catalysts of enormous power and high specificity.Consider a lump of sugar.It is combustible but quite difficult to set alight.A chemical catalyst would speed up itscomb

33、ustion,and we would end up with heat,a little light,carbon dioxide,and water.Swallowed and digested,the sucrose is broken down in many steps to carbon dioxide andP1:GIGWY001-11WY001-Bolsover-v2.clsSeptember 15,200321:31242HOW PROTEINS WORKwater by the action of at least 22 different enzymes,and the

34、energy released is used to driveother reactions in the body.At a basic level,a reaction carried out by an enzyme can be expressed asE+S?ES?EP?E+Pwhere E is the enzyme,which binds the substrate S to form the complex ES.The termsubstrate is used for a ligand that binds to an enzyme and that is then tr

35、ansformed to theproduct,P.The region of the protein where the substrate binds and the reaction occurs iscalled the active site.This binding is specific,often highly so.The enzyme-galactosidase(page 109)is moderately specific and will split not only lactose but also any other dis-accharide that has a

36、 glycosidic bond to-galactose.By contrast glycogen phosphorylasekinase(page 305)acts with absolute specificity on a single substrate,another enzymecalled glycogen phosphorylasenone of the thousands of other proteins in the cell cansubstitute.In general,the specificity of an enzyme is conferred by th

37、e shape of the activesite and by particular amino acid side chains that interact with the substrate.Binding of thesubstrate produces the enzymesubstrate complex ES;the catalytic function of the proteinthen converts the substrate to product,still bound to the enzyme in the complex EP.Finallythe produ

38、ct dissociates from the enzyme.Chemical engineers use catalysts made of manymaterials,and within cells there are catalysts called ribozymes that are made of RNA.However,only proteins,with their enormous repertoire of different shapes can producecatalysts of high selectivity.The catalytic rate consta

39、nt kcat(also known as the turnover number)of an enzymegives us an idea of the enormous catalytic power of most enzymes.It is defined as thenumber of molecules of substrate converted to product per molecule of enzyme per unittime(equally it is moles of substrate converted per mole of enzyme per unit

40、time).Manyenzymeshavekcatvaluesaround1000to10,000persecond.Thereciprocalofkcatisthetimetaken for a single event.Thus if kcatis 10,000 s1,one substrate molecule will be convertedevery tenth of a millisecond.Some enzymes achieve very much higher rates.Catalase,anenzyme found in peroxisomes,has a kcato

41、f 4 107s1,and so it takes only 25 ns to split amolecule of hydrogen peroxide into oxygen and water.The Initial Velocity of an Enzyme ReactionThe basic experiment in the study of enzyme behavior is measurement of the appearance ofproduct as a function of time(Fig.11.5).This is often called an enzyme

42、assay.The productappears most rapidly at the very beginning of the reaction.As the reaction progresses,therateatwhichtheproductappearsslowsdownandeventuallybecomeszerowhenthesystemhas reached equilibrium.All reactions are in principle reversible;thus the observed overallrate of the reaction is actua

43、lly the difference between the rate at which the product is beingformedandtherateatwhichtheproductisbeingbrokendownagaininthereversereaction.In many enzyme reactions the equilibrium lies strongly toward the product.fIN DEPTH 11.1 What to Measure in an Enzyme AssayIn an enzyme assay we measure the ap

44、pearance of product as a function of time.In principle we could do this by starting identical reactions in a series of test tubesand stopping the reaction in each test tube at a different time after the start andthen measuring the amount of product in each tube.P1:GIGWY001-11WY001-Bolsover-v2.clsSep

45、tember 15,200321:31ENZYMES ARE PROTEIN CATALYSTS243However,if either the substrate or the product has a property that can bemeasuredwhilethereactionisproceeding,thenthewholeassaycanbeperformedin one test tube.Many enzyme assays are done by using changes in the absorptionoflightwhenthesubstrateisconv

46、ertedtoproduct.Otheropticalpropertiescanbeused.In their original work Michaelis and Menten studied an enzyme that breaksthe disaccharide sucrose down into glucose and fructose.The mixture of glucoseplus fructose rotated polarized light differently from sucrose,and they used thisproperty to follow th

47、e course of the reaction.If there is no convenient opticalproperty,then others may be available;for example,one can monitor the progressof a reaction in which the polysaccharide glycogen(page 30)is hydrolyzed bydigestive enzymes by measuring the resulting fall in viscosity.We can simplify the analys

48、is of enzyme reactions if we consider only the start of thereaction,when there is no product present and so the back reaction can be neglected.Therate of reaction at time zero(the initial velocity v0,sometimes called the initial rate)isfound by plotting a graph of product concentration as a function

49、 of time and measuring theslope at time zero(Fig.11.5).In practice the slope is measured over the first 5%of the totalreaction.Initialvelocity,v0,isconvenientlyexpressedastherateatwhichtheconcentrationof the product increases,that is,moles per liter per second.Enzymes must be assayed under controlle

50、d conditions because temperature,pH,andother factors alter the activity.Most are such very effective catalysts that they must beassayed under conditions where the concentration of the enzyme is always very much lessthan the concentration of the substrate.Otherwise the reaction would be over in a fra