LEC9-11: Enzymes & Enzyme Kinetics Flashcards Preview

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Flashcards in LEC9-11: Enzymes & Enzyme Kinetics Deck (80):
1

what does it mean that biochemical reactions in the body are thermodynamically favored?

free energy (G) of the products is lower than that of the reactants 

aka ΔG has a negative value

2

if combined free energy of X+Y is less than that of A+B, which way is this reaction favored? 

 

A+B --> X+Y

favored in direction of X+Y 

3

what is the formula for Keq of the reaction A + B --> X + Y?

when is this reaction thermodynamically favored and spontaneously occurring?

Keq = [X][Y] / [A][B]

 

thermodynamically favored & spontaneously occurring when Keq < 1

4

what are transition states? what do they do to rates of rxns? how are they surpassed?

transition states: high-energy intermediates that are barriers to spontaneous reaction occurring

cause highly favorable reactions to proceed at very low rates spontaneously

energy required to each one is G+, its activation energy

 

5

how do enzymes interact with reactants in a chem rxn? what is the consequence?

enzymes breifly bond w/ reactants (substrates) in their transition states at the active site of an enzyme

these reactant-enzyme complexes have low free energy compared to transition state of non-catalyzed reaction 

thus enzymes accelerate the reaction

 

6

how does the enzyme chymotrypsin work?

lowers the transition states' energy in the rxn btwn H2O + a polypeptide 

1st peak: energy to strip peptide of water molecules in the aq environment, allows initial bond form btwn substrate and enzyme 

this bond forms, results in drop in energy

2nd peak: first of 2 transition states catalyzed by chymotropin

3rd, tallest peak: maximum energy, assoc w/ 2nd transition state

 

7

with an enzyme present, what changes - the activation energy, or the net energy? how is change?

with the enzyme present,

activation energy for catalyzed reaction is smaller than for the spontaneous reaction so reaction occurs,

even though 

net energy change is the same for catalyzed and spontaneous reaction

8

what is this curve showing

 

Q image thumb

effect of enzyme-mediated catalysis on energy profile of a reaction: 

enzyme lowers the transition states for the reaction 

net energy change remains the same for catalyzed and spontaneous reaction

9

do enzymes impact equilibrium of reaction? 

why/why not?

enzymes DO NOT affect equilibrium of the reaction 

equilibrium depends solely on ΔG

ENZYME CHANGES ONLY THE REACTION RATE

10

what is the enzyme dogma?

to what extent does it occur?

ENZYME ONLY CHANGES THE REACTION RATE

THEY DO NO AFFECT THE EQUILIBRIUM OF THE REACTION

however, this change can be so dramatic that it can be like an "off" to "on" switch for a rxn: 

usu enzymes increase rate by factor of 106-1014

a rxn that'd spontaneously occur 1x/yr could, w/ enzyme catalysis, occur 30x/millisecond (1011-fold increase)

11

what would happen in this reaction if B is continually depleted? 

 

Q image thumb

rate of A --> B increases 

per Le Chatelier's principle (change in concentration of a reactant or product will shift equilibrium of reactions involved) 

12

what is a "uni-uni" reaction

rxn in which a single reactant (substrate) transformed into single product 

i.e. when an isomerase catalyzes the conversion btwn stereoisomers

13

what is a "bi-bi" reaction?

i.e. phosphotransferase, transfers a phosphate group from 1 substrate to another 

2 substrates, 2 products

14

what is a "bi-uni" reaction?

2 substrates joining together into 1 product 

i.e. by a ligase

15

where is an enzyme's active site? 

what happens there? 

enzyme binds substrate in its active site

active site lies in a groove or pocket of the enzyme, typically includes residues from different segments of the polypeptide chain

16

what kind of reaction occurs between chymotrypsin and phenylalanine?

bi-bi

17

what is the binding site of the lock-and-key model?

when the binding site is optimized for the substrate 

there's a relatively stable region of an enzyme for its corresponding reactant 

recognizes the reactant based on characteristics like its topology, electrostatic profile, potential to form bonds w/ amino acids in the active site 

 

18

what is the active site in the induced-fit model?

unoccupied binding site has low affinity for the substrate, but binding induces a conformational change in enzyme that makes active site have a high affinity for substrate

this brings reactive groups of enzyme close to substrate

may be only one transition state but may need series of transition states to be induced before reaction's complete

more accurate model of enzyme-substrate binding

19

in a thermodynamically favorable reaction, what is the relationship btwn energy of the reactants and energy of the products? 

what must be achieved to get from reactants to products?

energy of the reactants is HIGHER than energy of the products 

need activation energy to attain the transition state, when bond has been made but not cleaved; transition state energy > reactant energy

20

how does the substrate react to its enzyme in the induced-fit model?

requires substrate to undergo steric changes that bring it closer to a transition state 

often, there are several transition state induced before rxn is complete 

these conformational changes of enzyme bring other regions of the protein close to the substrate; they interact, or places multiple subtrates near each other in an orientation to favor a rxn

21

describe the relationship between glucose and glucokinase 

what kind of enzyme-substrate binding is this?

induced-fit model 

glucokinase (a hexokinase) catalyzes the phosphorylation of glucose 

glucokinase has 2 domains, joined by a hinge region

when unbound, glucokinase is open conformation

when glucose binds, glucokinase is closed, & thus catalytically active 

22

why does the induced fit model make more thermodynamic sense than the lock and key model?

lock-and-key provides a large decrease in G upon binding (ES), but reaching transition state from this low energy would be rare

induced fit, have small decrease in G upon binding (ES), but reaching transition state is greatly reduced compared to spontaneous, b/c enzyme-transition state complex has much lower G than transition state alone. reaction proceeds more rapidly. 

23

what is enzyme kinetics 

the study of the rate of enzymatically catalyzed rxns as a fxn of variables that include substrate & enzyme concentration, drugs that inhibit enzyme fxn, temperature, pH, etc.

24

how is the rate of rxn of substrate --> product measured?

measured when the rxn is initiated, by adding substrate to enzyme 

measure conversion of substrate --> product before product accumulates & reverse rxn becomes significant 

 

25

what is the inital rate of a substrate --> product rxn called?

initial velocity, V0

26

what does this show? explain!

 

Q image thumb

for most enzymes, V0, initial velocity, is a hyperbolic function of [S]

when [S] is low, V0 is proportional to [S] = first-order kinetics

when [S] is high, Vreaches asymptote, Vmax and becomes independent of [S] = zero-order kinetics

27

what is first-order vs. zero-order reaction w/ enzyme-substrate relationship?

first-order: when V0, initial rate, is directly proportional to [S]

zero-order: when [S] is very high, rate is independent of [S]; relationship flattens, and V0 becomes constant 

 

28

what is Vmax?

highest possible reaction rate for a given catalyzed reaction 

29

what is Km

what does it mean if the reaction's Km is lower w/ 1 enzyme than another? 

Km = substrate concentration at which V0 is 50% of Vmax 

aka affinity of the substrate for the enzyme 

lower Km = higher affinity; higher Km = lower affinity

30

which enzyme has a lower Km, a higher affinity, and what is the relationship of their Vmax?

Q image thumb

purple: lower Km so higher affinity substrate 

same Vmax though of both substrates w/ the enzyme, even though affinities differ

31

what is the michaelis-menten equation?

describes hyperbolic relationship between [S] and Km of enzyme-substrate 

Km is the Michaelis constant

if [S] < Km, V0 is directly proportional to [S] = first-order behavior

if [S] > Km, V0 = Vmax = zero-order behavior

A image thumb
32

state the lineweaver-burke transformatin of the michaelis-mentin equation 

name the variables

 

slope = Km / Vmax 

y-intercept = 1/Vmax

 

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33

how does a competitive inhibitor work?

what is its apparent effect on K​and on Vmax?

competes for the same binding site as the substrate 

increases Km

no effect on Vmax

34

what is the M-M equation w/ a competitive inhibitor?

A image thumb
35

how does a noncompetitive inhibitor work?

what happens to Km?

what happens to Vmax

doesn't bind at substrate-binding site, so cannot be displaced by increase [S] 

inhibitor allows substrate to interact normally w/ active site, but interferes w/ enzyme's ability to catalyze rxn 

Vmax is reduced (higher on Y-axis)

Km is unchanged (same on X-axis)

 

36

how does an uncompetitive inhibitor work?

what happens to Vmax and Km? 

binds when the substrate is bound, b/c ES complex creates a binding site for the inhibitor

Vmax and Km both are reduced

37

with irreversible inhibition, what kind of binding does inhibitor do? 

what happens to Vmax and Km

covalent, irreversible bonding of inhibitor-enzyme 

decreases Vmax 

Km is unchanged 

38

what are the effects of irreversible inhibition?

1) as duration of exposure to inhibitor increases, so does the number of inhibited enzyme molecules 

2) even after inhibitor has been removed, enzyme remains inhibited (i.e. if drug is an irreversible inhibitor, must synthesize new enzyme to restore enzymatic function)

39

what is an effector? what kinds are there?

usually a molecule that's part of a pathway 

binds noncovalently to a subunit of a regulatory enzyme, induces a change in affinity of substrate for binding site OR alters enzymatic efficiency of active site 

can be nevative or positive

 

40

what are homotropic and heterotropic modulation?

homotropic modulation: what the substrate itself is the effector; enzyme has >1 catalytic site, and binding of 1st site alters substrate affinity of remaining sites 

heterotropic modulation: if effector is a molecule other than a substrate

41

what are allosteric enzymes?

enzymes that're subject to regulation by effectors 

often are comprised of multiple subunits 

42

what is alpha-ALA synthase an example of?

an allosteric enzyme

its activity is under the control of an effecotr (heme)

43

what is aspartate carbamoyltransferase an example of?

a multimeric regulatory enzyme

44

what is positive cooperativity? what does it look like on a graph? 

what is it an example of?

homotropic modulation 

1st site's occupation increases affinity of subsequent sites to bind

V0 vs [S] is sigmoidal (i.e. hemoglobin)

 

A image thumb
45

describe model of homotropic allostery

enzyme is a homomer of 2 subunits, each can either be in relaxed or taut state

R=high affinity; T=low 

both subunits initially are in T 

when substrate binds 1 subunit, it induces a fit/creates a high-affinity site, converts that subunit to the R conformation 

now other subunit also assumes R conformation, presenting a high-affinity binding site 

 

A image thumb
46

what is unique about glucokinase re: positive cooperativity? 

glucokinase has only 1 binding site glucose 

enzyme assumes more open conformation upon binding a molecule of glucose, exposing active site 

once glucose is phosphorylated and exits active site, the open conformation is retained briefly 

this increases access to active site for subsequent glucose molecules 

47

how does positive heterotropic modulation work? 

binding to a site on a regulatory subunit (R) increases affinity of catalytic subunit (S) for substrate 

effectors here don't covalently modify the enzyme - they bind, induce conformational change, and dissociate

usually binding molecule is the rate limiting step for the pathway 

A image thumb
48

what is heme synthesis pathway an example of? explain 

negative heterotropic modulation 

heme inhibits g-ALA synthase, 7 steps above it in heme synthesis pathway 

shows end-product inhibition/feedback inhibition 

it catalyzes the rate limiting step, keeps level of activity in the pathway within narrow phsiological range

49

what is heterotropic effectors' impact on Km and Vmax?

activators (positive effectors): decrease Km, no effect on Vmax 

negative effectors: increase Km, no effect on Vmax (apparently competitive inhibition) OR Km unaffected, Vmax reduced (apparently non-competitive inhibition)

50

where do heterotropic effectors come from, re: signaling pathway where their effected allosteric enzyme is? 

therefore what is their function?

come from outside the signaling pathway 

therefore don't maintain homeostatic control, instead serve signaling role 

51

what is PKA activation by cAMP example of?

positive heterotropic modulator 

cAMP binding induces dissociation of catalytic and regulatory subunits of PKA 

therefore catalytic subunits can phosphorylate their substrates 

52

what is covalent modification 

 

addition of a modifying group to a regulatory enzyme 

particularly occurs w/ Tyr, Ser, Thr, which're substrates for phsophorylation by various protein kinases 

once attach fxn group, enzymatic activity req'd to remove them 

THOSE enzymes that add/remove active groups often are subj to regulation; therefore, whole process needs info from variety of metabolic, signaling pathways 

53

what is most common form of covalent modification? what accomplishes this or undoes it?

phosphorylation/dephosphorylation 

protein kinases: add phosphate group

protein phosphatases: catalyzes hydrolysis of phosphate group

54

is one protein regulated by one protein kinase?

no, often have many phosphorylation sites on a typical protein 

a particular protein may have overlapping phosphorylation sites for different protein kinases 

55

what is a consensus sequence?

sequence that a protein kinase/any enzyme that produces a covalent modification recognizes on its target protein

indicates that that protein has a pohsphorylation site for that kinase

56

what does Ca2+/Calmodulin-depednent protein kinase II (CaMKII) demonstrate? 

describe binding process 

positive cooperativity, Ca2+ is a positive effector of calmodulin:

Ca2+ binds calmodulin, which has 4 Ca2+ binding sites

when 4 sites are bound, calmodulin undergoes conformational change from closed --> open

affinity of Ca2+ for calmodulin increases as binding sites fill up 

when all 4 Ca2+ binding sites are filled, calmodulin is activated, can bind CaMKII 

 

57

what happens when calmodulin binds CaMKII? 

usually, CaMKII's catalytic region is blocked by an inhibitory/"pseudosubstrate" region of the protein 

when calmodulin binds, conformation cahgne in CaMKII moves inhibitory region away from active site 

active site now accessible to substrates for CaMKII

1 substrate=itself; CaMKII phosphorylates itself, via autophosphorylation, prevents itself from returning to closed conformation even when Ca2+ levels decline and calmodulin dissociates from CaMKII = positive homotropic effector that acts on itself

eventually, protein phosphatase 1 removes phosphate, stops CaMKII activity

58

why does CamKII have a "memory"?

CaMKII is positive homotropic effector that acts on itslef by autophosphorylating & therefore keeping its active site open to phosphorylate other enzymes

it "remembers" the Ca2+ signal long after binding occurred

plays key role in formation & retention of memories in the brain 

eventually, protein phosphatase 1 removes phosphate from CaMKII, terminates its activity 

59

what are zymogens?

an inactive precursor that, when proteolysed, liberates an enzyme

60

what are trypsinogen and chymotrypsinogen? where do they come from, what do they do?

zymogens secreted by the pancreas into the duodenum, involved in digestion

contian sequences for trypsin and chymotripsin 

once in duodenum, they encounter enteropeptidase, a locally-secreted enzyme which cleaves tripsin from trypsinogen & trypsin in turn liberates chymotrypsin from its zymogen

61

how is the clotting pathway regulated? describe it

clotting factors are proteases that liberate enzymes that particiapte in blood coagulation 

for clotting to occur, proteins that coagulate must be liberated from their zymogens 

when they kick off, they rapidly induce clotting 

at each step, an active protease is generated, which catalyzes the next zymogen --> active enzyme rxn

is a very highly regulated pathway so clotting cascade doesn't activate in response to an aberrant signal 

62

what is an enzyme cofactor? 

what is its function?

many enzymes require an add'l small molecule, cofactor, to peform their catalytic fxn

sometimes the cofactor is covalently bound to the protein part of the enzyme = apoenzyme, full complex = holoenzyme

focator can be a metal ion (Fe2+, Cu2+) or organic molecule = coenzyme, (vitamin or derived from vitamin)

they transfer active groups from 1 substrate to another; do re-dox rxns; are generated for reuse btwn reactions

63

examples of important enzyme cofactors?

A image thumb
64

what are isoenzymes?

what does their existence demonstrate?

multiple enzymes that perform the same reaction

demonstrates that the same rxn might require different regulation, or different substrate concentrations, in 1 cell type or tissue than in another

65

what are the hexokinases' function? what are they an example of?

hexokinases are 4 isozymes - hexokinase I, II, III, IV - that phosphorylate simple sugars 

hexokinase IV = glucokinase, is diff from the other isoforms,

66

what makes glucokinase unique from the other hexokinase isoforms?

it uses excess glucose for glycogenesis & glycolysis

1) it is a monomer, the others are dimers 

2) it is not inhibited by its product glucose-6-phosphate under physiologic conditions

3) it has a much higher Km for glucose than the other hexokinases 

4) it is abundant in the liver, not ubiquitous throughout the tissues like the other hexokinases 

 

67

what does glucokinases's high Km mean re: its relationship to glucose?

b/c glucokinase is abundant in liver, its main role is to use excess glucose for glycogenesis and glycolysis 

b/c no feedback inhibition, can do this even if G6P accumulates in liver cells 

oppositely, if glucose is scarce, high Km means that glucokinase assures that available clucose is used by hexokinases in other tissues (ie brain) to maintain metabolic processes

68

what is creatine kinase?

where are its isozymes found, and why are its isozymes clinically significant?

CK is enzyme in energy metabolism, found mostly in muscle 

CK is dimer of M and/or B subunits, has 3 isozymes: 

1) CK1: tissues, including brain

2) CK2: heart 

3) CK3: skeeltal muscle, heart 

normally, blood is almost all CK3 b/c of normal turnover of skeletal muscle; but increase in CK2 in blood is diagnostic of MYOCARDIAL INFARCTION in a pt who has chest pain 

69

what is kcat? what does it measure?

the turnover number of an enzyme: the number of operations that a single molecule of an enzyme can perform per second when the enzyme is saturated

allows us to compare the efficiencies of different enzymes

 

 

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70

what is the specificity constant? what does it explain?

what does a large / a small value indicate?

Kcat/Km

allows us to compare enzyme efficiency under physiological conditions

indiciates how effecient the enzyme is when free binding sites are abdundant  

if large: reaction can proceed at high rate even if [S] is low or enzyme isn't highly expressed

71

what is catalytic perfection?

Kcat/Km must be between 108 and 109 M-1s-1

if an enzyme's Kcat/Km is close to this, enzyme is catalytically perfect 

boundaries exist b/c diffusion rate of molecules in aqueous solution dtermines the minimum time req'd for binding sites to be vacated and reoccupied  

 

72

what are the 6 categories of enzymes?

oxidoreductases 

transferases 

hydrolasese

lyases

isomerases

ligases

73

what do oxidoreductases do

transfer electrons (hydride ions or H atoms)

74

what do transferases do

group-transfer reactions

75

what do hydrolases do

hydrolysis reactions (transfer of functional groups to water)

76

what do lyases do

addition of groups to double bonds, or formation of double bonds by removal of groups

77

what do isomerases do

transfer of groups within molecules to yield isomeric forms

78

what do ligases do

formation of C-C, C-S, C-O, C-N bonds by condensation reactions coupled to ATP cleavage

79

are all enzymes proteins? 

no

RNAs that catalyze reactions, ribozymes, exist 

i.e. ribosomal RNAs (rRNA) catalyze many steps of protein synthesis 

much more ancient than enzymes

ribozymes are not subj to high degree of regulation as in many protein enzymes

80

what's the most important/complex ribozyme?

the ribosome 

where mRNA translation happens, almost entirely by ribosomal RNA, w/ ribosomal proteins playing only supportive role 

 

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