28 Pages
Unlock Document

University of Toronto Scarborough
Biological Sciences
Shelley A.Brunt

lec09 mechanisms of enzyme action continued 1. http://higheredbcs.wiley.com/legacy/college/voet/0470129301/guided_exp/guided_exploratio n_11/michaelis_menten.html a. click link for a review of michaelis menten kinetics mechanisms of enzyme action 2. look at how enzymes catalyze reactions 3. look at reactants, intermediates, and products 4. specific role of particular amino acids in the active sites of an enzyme 5. remember enzymatic enzymes require movement of electrons just as in chemical reactions 6. profs notes a. looked at how enzymes catalyze reactions b. continue by talking about different mechanisms enzymes use c. expand on concept when substrate and enzyme come together d. importance of reactants and intermediates and transition states e. concentration on few enzymes and the role of specific amino acids f. enzymatically, enzymes require movement of electrons as in chemical reactions active site events in enzymes 1. a look at reaction mechanisms a. when considering the mode of action of an enzyme i. the following are important 1. which amino acid residues catalyze reaction at active site 2. spatial relationship of reactive amino acids 3. mechanisms by which reactive amino acids catalyze reaction 4. enzymes catalyze chemical reactions in many ways a. but all reactions have in common the requirement that some reactive group on the enzyme interacts with the substrate 5. side chain reactive groups are the ones involved in the action of the enzymes a. hydrocarbon side chains not usually reactive b. reactive groups include i. imidazole group of histidine ii. hydroxyl group of serine iii. carboxyl side chains of aspartate and glutamate iv. sulfhydryl group of cysteine v. amino side chain of lysine vi. phenol group of tyrosine 6. formation of an enzyme substrate complex places reactants in proximity to reactive residues in the enzyme active site a. ionizable side chains participate in the two major chemical modes of catalysis 7. prof notes a. modes enzymes might use i. how to determine what mode is being used ii. what amino acids are being used b. important is spatial interaction i. e.g. aspartate to glutamate 1. different length of side chain 2. alters stereochemistry in terms of length of interaction 3. significant disability to function ii. e.g. serine vs threonine 1. difference in length of side chain iii. e.g. arginine 1. rare occurrence because of high pI c. what are mechanisms by which amino acids catalyze reactions? i. chemical modes 1. acid base 2. covalent ii. binding modes 1. proximity 2. transition state stabilization d. rest of the amino acids i. provide structure to active site e. enzyme reactions have a reactive group in common that interacts with the substrates f. the most important reactive amino acid in enzymes is histidine i. pI is near neutral pH ii. able to function efficiently at body temperature g. formation of enzyme substrate complex puts enzyme in close proximity i. proximity of substrate to enzyme is important nucleophilic substitutions 1. shown by electron pushing diagrams a. covalent bonds are broken and / or new covalent bond formed i. tetrahedral intermediate that may be stable enough to be detected ii. ionic intermediates include: 1. electron rich (nucleophilic) a. nucleophile has a negative charge or unshared electron pair b. nucleophiles attack electrophiles in a process called a nucleophilic attack i. i.e. substitution ii. e.g. serine proteases 2. electron poor (electrophilic) 2. nucleophilic substitutions a. nucleophilic attack i. creation of tetrahedral intermediate ii. creation of product + leaving group b. intermediate i. by definition is detectable ii. you can find it during the process c. transition states i. are not detectable ii. you cannot find and isolate transition states d. electrophilic i. electron poor ii. loves electrons e. nucleophilic i. electron rich nucleophilic substitution through direct replacement 1. attacking group is directly added to the central atom a. directly opposite to the leaving group b. forms a transition state i. carbon maximally binds 4 groups ii. transition state has 5 groups 1. therefore unstable 2. high energy state whose structure is between the reactant and product 3. i.e. attack and leaving occurs simultaneously 2. direct replacement a. nucleophilic attack i. formation of unstable transition state ii. nucleophilic attack opposite of leaving group 1. causes leaving group to leave b. key point i. formation of transition state (unstable) instead of intermediate (stable) ii. attack and exit occur almost simultaneously 1. i.e. cannot isolate transition state cleavage reactions 1. covalent bonds can be cleaved in two ways a. both electrons stay with one atom i. most common b. one electron can remain with each atom i. cleavage of a C-H bond almost always produces two ions c. carbanion i. when the carbon atoms retain both electrons d. carbocation i. when the carbom atom loses both electrons e. free radicals i. when one electron remains with each product ii. molecule with an unpaired electron iii. usually unstable iv. formation of free radicals are highly caustic to cellular function 1. extra reactive -- react with anything 2. damage cells oxidation-reduction reactions 1. critical for supplying biological energy 2. transfer of electrons from one species to another 3. oil rig a. oxidation is loss of electrons b. reduction is gain of electrons 4. redox a. reactions always occur together b. one substrate is oxidized c. one is reduced 5. oxidizing agent a. causes oxidation b. takes electrons from substrate being oxidized c. the agent itself becomes reduced 6. reducing agent a. causes reduction b. the agent itself becomes oxidized 7. oxidation a. e.g. i. removal of hydrogen 1. most common ii. addition of oxygen iii. removal of electrons 8. redox reactions carried out by enzyme class oxidoreductases a. subclasses i. dehydrogenations 1. dehydrogenases a. cleaving C-H bonds b. creating hydride ion c. accompanied by reduction where another substrate gains electrons by reacting with the hydride ion 9. prof notes a. co-enzymes and co-substrates that accept electrons and undergo redox reactions i. complicate things ii. talk more about this later on iii. for now we assume only enzyme and substrate b. most common oxidation is the removal of hydrogen c. ... progress of the reaction 1. transition state and intermediates during catalysis a. transition state i. unstable (half short life) ii. occurs at the peak of the activation barrier b. activation energy i. the lower the barrier the more stable the transition state and the more likely the reaction proceeds 2. graph a. x axis i. course of reaction ii. reaction coordinate iii. progress of bond breaking proceeds to the right b. y axis i. free energy c. do not need to know how to draw enzyme catalysis diagrams 3. prof notes a. what is happening when substrate brought close to enzyme i. formation of unstable transition state 1. always occurs at peak of activation barrier a. from substrate to product i. difference in free energy 1. the lower the barrier, the easier the forward reaction is facilitated catalysts 1. catalysts (enzymes) a. lower activation energies b. participate in reactions by stabilizing transition states c. accelerate reactions by lowering the overall activation energy energy diagram for a reaction with intermediates 2. intermediates unlike transition states can be stable enough to be detected 3. prof notes a. two transition states in this graph with an intermediate b. intermediate i. meta stable state c. if intermediate does not form, reaction will not go forward d. note the two different barriers for the two transition states enzymatic catalysts lower the energy needed 1. effect of reactants being bound by enzyme a. lowers activation energy by increasing formation of transition state i. i.e. brings reactants together in substrate binding site 2. effect of reactants and transition state being bound by enzyme a. lower activation further b. complex with enzyme and transition state stabilizing the transition state 3. prof notes a. enzymes lower activation energy by increasing the formation of transition state b. brings them close together so that they can react better i. importance of proximity c. enzyme is potentiating the reaction by bringing reactions in close proximity i. what does potentiating mean? 1. potential to go forward catalytic mechanisms 1. sometimes enzymes use multiple catalytic mechanisms a. acid base catalysis b. covalent catalysis c. metal ion catalysis d. proximity and orientation effects e. preferential binding of transition state chemical modes of enzymatic catalysis 2. the formation of ES complexes assist reactions by placing reactants close to the reactive amino acid residues of the active site of the enzyme 3. ionizable side chains can take part in two kinds of chemical catalysis a. acid base catalysis b. covalent catalysis 4. prof notes a. not just about getting substrate to active site i. it is about whether the substrate fits well in the active site 1. e.g. difference between aspartate and glutamate a. different length of side chains active sites 1. most active sites are lined by hydrophobic amino acid residues a. there are a few polar, ionizable residues as well 2. polar amino acid residues on enzyme (or coenzymes on occasion) undergo chemical change during enzymatic catalysis a. why? i. acid-base b. what did she emphasize about this point? 3. what do we know about ionizable groups? a. what would happen if water entered the active site? i. dissociation of ions 1. cannot interact in active site a. therefore don't want water present in active site i. need to exclude water 1. therefore major players in active site are primarily hydrophobic amino acids a. to create hydrophobic pockets in the active site to exclude water 4. today a. not talking about co-substrates and co-enzymes i. only talking about enzymes and substrates types of ionizable residues found in the active sites of enzymes 1. catalytic functions of reactive groups of ionizable amino acids a. table on slide 15 i. do we need to know the principal functions of each amino acid? 1. aspartate a. cation binding b. negatively charged 2. glutamate a. cation binding b. negatively charged 3. histidine a. acceptor or donor b. functions at neutral pH 4. cysteine a. covalent binding of acyl groups b. disulfide bridges 5. tyrosine a. hydrogen bonding to ligands b. phenol group 6. lysine a. anion binding b. positively charged 7. arginine a. anion binding b. positively charged 8. serine a. covalent binding of acyl groups b. hydroxyl group 2. prof notes a. kind of reactions that take place b. histidine is workhorse of enzymes i. has acceptor and donor c. tyrosines unique ability to interact with phosphate groups d. difference between lysine and arginine i. one is different in terms of pI ii. therefore able to interact in terms of proton transfer 1. on top of regular function of anion binding pKa values of ionizable groups of amino acid residues in proteins can differ than in free amino acids 1. key point a. amino acid residues have may have different pKa values than a single amino acid by itself 2. slide 16 a. table 6.2 i. typical pKa values of ionizable groups of amino acids in proteins b. table 3.2 i. pKa values of acidic and basic constituent amino acids at 25 degrees pKa values within a protein 1. microenvironment can change the pKa values within a protein compared to free amino acids 2. pH can affect the reaction rate 3. remember only a small number of amino acid residues take part directly in catalyzing reactions a. i.e. small portion of protein interacts with the active site of enzymes i. most contribute indirectly by maintaining tertiary structure ii. enzymes can have 2 to 6 key catalytic residues 4. prof notes a. microenvironment can change pKa within a protein i. e.g. glutamate pI normally around 3 1. localized environment may shift pI to 5 b. because of localized microenvironment i. pKa can change c. because of relying on reactive amino acids i. pI becomes important in how they function 1. if outside their range of functioning, they DONT function a. e.g. enzymes functioning in stomach must function in low pH i. if active site alters tertiary structure 1. substrate may not be able to interact a. substrate either fits better or not as well with the changing conformation of the active site most common residues in catalytic sites 1. average protein a. histidine is relatively rare protein compared to other amino acids i. but highly common in catalytic residues 1. i.e. common at active sites 2. whats the difference between aspartate and glutamate a. who has shorter side chain? i. aspartate 1. allows closer proximity between substrate and enzyme a. preferred tighter binding 3. in terms of confines of active sites a. amino acids that populates active sites may not be very populated in terms of the protein itself roles of amino acid residues 1. roles of amino acid residues not directly involved in catalysis a. is to assist or enhance the catalytic residues i. catalytic residues in addition to being directly involved in catalysis can also be involved in 1. substrate binding 2. stabilization of transition state 3. interaction with cofactors 2. prof notes a. amino acid residues key for i. getting substrates bound ii. stabilization of transition states acid base catalysis 1. increased reaction rate is accomplished by the catalytic transfer of a proton 2. one of the most common form of catalysis in enzymatic reactions 3. enzymes that carry this out rely on side chains that can donate and accept protons under the near neutral pH of the cell 4. general acid base catalysis vs specific acid base catalysis a. specific i. catalysis by H+ or OH- b. the active site of the enzymes are providing the biological solution of acid or bases i. missed what she emphasized about this point 1. region where acid base takes place 5. histidine which has an imidazole / imidazolium side chain has a pKa of 6-7 in most proteins a. provides the ideal group for proton transfer at neutral pH b. it is a common catalytic residue 6. prof notes a. transfer of proteins b. most likely to see histidines in this type of catalysis c. general vs specific acid base catalysis i. what is the difference? 1. refer to point above d. histidine i. imidazole side chain 1. pKa of 6-7 ii. ideal group for protein transfer 7. slide 21 a. a proton acceptor (B) can cleave OH NH or CH bonds by removing a proton b. proton acceptor (B) can also take part in the cleavage of other bonds involving carbons such as CN i. by generation of OH in neutral solution by the removal of a proton from water c. prof notes i. protein acceptor 1. cleaves OH / NH / CH groups by removing a proton 2. final result a. B enters (proton acceptor) b. X leaves (leaving group) ii. B can also take part in cleavage of other bonds involving water 1. forming OH by removal of proton 2. not really certain about this point covalent catalysis 1. in this reaction a. a substrate is bound covalently to the enzyme forming a reactive intermediate b. the reacting side chain is either a nucleophile or an electrophile i. nucleophilic catalysis is more common c. in the second part of the reaction i. a part of the substrate is transferred from the intermediate to a second substrate ii. common mechanism for coupling different reactions 1. A-X + E  X-E + A 2. X-E + B  B-X + E a. group X has been transferred from substrate A to B in a two step process via the covalent ES complex E-X i. concept: 1. the ability to couple reactions is an important property of an enzyme 2. prof notes a. substrate bound covalently to enzyme at some point in interaction i. creation of stable enzyme substrate complex intermediate b. reactive side chain can either be electrophilic or nucleophilic i. though nucleophilic catalysis is more common c. transfer from intermediate to second substrate i. A-X + E  X-E + A ii. X-E + B  B-X + E d. key point i. ability to couple reactions ii. important property of enzyme covalent catalysis  ping pong model of enzyme kinetics 1. example of group transfer by covalent catalysis a. e.g. sucrose (glucose and fructose) + Pi  glucose-1-phosphate + fructose i. breaking the reaction down: two step process 1. sucrose + enzyme  glucosyl-enzyme (ES intermediate) + fructose 2. glucosyl-enzyme (can donate the glucose unit to phosphate) + Pi (B - proton acceptor)  glucose-1-phosphate + enzyme ii. alternatively 1. the ES intermediate can donate it to fructose (which is A in the reaction) in the reverse reaction 2. remember ping pong kinetics is an indicator of covalent catalysis a. if you can identify (i.e. detect the intermediate) that is being altered in a reaction i. this is also evidence of a covalent catalysis 3. prof notes a. example of ping pong kinetics  covalent catalysis b. identification of intermediate that is being altered in reaction i. indication of covalent catalysis taking place pH can affect the rate of enzyme reaction 1. slide 24 a. shows profile of enzyme papain i. to the left and right of the curve you have titration of relevant side chains in the active site 1. i.e. enzyme is only active when in the form of a thiolate (deprotonated form -S-) and imidazolium (protonated histidine side chain pair) 2. how to you titrate the side chains? ii. cysteine is being deprotonated 1. perturbed pKa of 3.4 a. normally thiol groups in proteins have pKas of 8 to 9.5 iii. histidine is protonated 1. perturbed pKa of 8.3 a. normally 6-7 iv. result 1. histidine becomes deprotonated 2. enzyme cant function 3. bell shaped graph if two ionizable groups involved a. sigmoidal if one group involved b. prof notes i. whats going on in this reaction 1. right of curve a. titration of side chain 2. what do we know about normal pKa of side chain of cysteine? a. it is quite high i. why is it perturbed? 1. what does this mean? ii. enzyme is only active when 1. cysteine is in deprotonated form and 2. histidine is in protonated form iii. what did she say about hyperbolic bell shaped and sigmoidal curves? 1. what is active and what is not? a. bell shaped involves two ionizable groups b. sigmoidal involves on ion ionizable groups iv. talked really fast on this slide, didn't follow much of what she said v. environment does make a difference 1. once you put amino acid in active site a. change in the pKa at which amino acids normally function ionizable residues of papain 1. imidazole N is blue a. histidine is a proton donor in the functional active site 2. sulfur atom is yellow a. cysteine is nucleophilic 3. prof notes a. cysteine is nucleophilic b. histidine is proton donor schematic of papin side chains in active site 1. goes from a. both protonated b. inactive form 2. to a. cysteine deprotonated b. active form 3. to a. both deprotonated b. inactive form 4. prof notes a. as you move up pH range i. becomes active b. as you move up pH range further i. becomes inactive again activity of enzyme papain 1. inactive when both protonated 2. cysteine side chain becomes deprotonated as it transits through pH of 3.4 a. sulfur is not deprotonated 3. enzyme is active when there is a. ionic interaction between S-group and the protonated histidine 4. loss of the proton from the histidine as transit through pH of 8.3 a. leads to an inactive enzyme 5. prof notes a. inactive enzyme i. both protonated b. move of pH range i. scenario where pKa of cysteine is altered ii. shared proton between cysteine and histidine 1. both deprotonated c. substrate not present i. pH has effect on active sites ii. if pH is wrong, active amino acids will be in wrong form 1. i.e. wont react properly d. ... e. if you put enzyme in the wrong pH i. won't function diffusion controlled reactions 1. a reaction that occurs with every collision between reactant molecules 2. under physiological conditions 8 9 a. the rate of diffusion controlled reactions is about 10 to 10 per ms (kcat/km) 3. if there is an electrostatic attraction between reactants a. the frequency of the reaction can be increased b. e.g. see this in various enzymes i. catalase ii. superoxide dismutase iii. triose phosphate isomerase 1. all of these enzymes catalyze reactions that are so simple, that the rate determining steps are as fast as binding of substrates to the enzymes a. what was the emphasis on this point? 4. prof notes a. diffusion controlled reactions b. simplest and fastest reaction that takes place i. about collision between enzyme and substrate c. ability of enzyme and substrate to be brought together i. proximity d. electrostatic attraction between reactants i. frequency of reaction increases e. enzyme classes i. catalase ii. superoxide dismutates iii. triose phosphate isomerase triose phosphate isomerase 1. catalyzes the interconversion shown during glycolysis a. dihydroxyacetone phosphate (DHAP)  D-glyceraldehyde-3-phosphate (G3P) 2. changing position of double bond and moving OH group 3. how fast enzyme and substrate comes together is the rate determining step general acid-base catalysis mechanism 1. proposed for the reaction catalyzed by triose phosphate isom
More Less

Related notes for BIOC12H3

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.