LEC9-11: Enzymes & Enzyme Kinetics

Question 1

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

Answer 1

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

aka ΔG has a **negative **value

Question 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

Answer 2

favored in direction of X+Y

Question 3

what is the formula for K_eq of the reaction A + B –> X + Y?

when is this reaction thermodynamically favored and spontaneously occurring?

Answer 3

K_eq = [X][Y] / [A][B]

thermodynamically favored & spontaneously occurring when K_eq < 1

Question 4

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

Answer 4

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

Question 5

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

Answer 5

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

Question 6

how does the enzyme chymotrypsin work?

Answer 6

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

Question 7

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

Answer 7

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

Question 8

what is this curve showing

Answer 8

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

Question 9

do enzymes impact equilibrium of reaction?

why/why not?

Answer 9

enzymes DO NOT affect equilibrium of the reaction

equilibrium depends solely on ΔG

ENZYME CHANGES ONLY THE REACTION RATE

Question 10

what is the enzyme dogma?

to what extent does it occur?

Answer 10

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)

Question 11

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

Answer 11

rate of A –> B increases

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

Question 12

what is a “uni-uni” reaction

Answer 12

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

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

Question 13

what is a “bi-bi” reaction?

Answer 13

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

2 substrates, 2 products

Question 14

what is a “bi-uni” reaction?

Answer 14

2 substrates joining together into 1 product

i.e. by a ligase

Question 15

where is an enzyme’s active site?

what happens there?

Answer 15

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

Question 16

what kind of reaction occurs between chymotrypsin and phenylalanine?

Answer 16

bi-bi

Question 17

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

Answer 17

**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

Question 18

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

Answer 18

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

Question 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?

Answer 19

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

Question 20

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

Answer 20

**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

Question 21

describe the relationship between glucose and glucokinase

what kind of enzyme-substrate binding is this?

Answer 21

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 **

Question 22

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

Answer 22

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.

Question 23

what is enzyme kinetics

Answer 23

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.

Question 24

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

Answer 24

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

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

Question 25

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

Answer 25

initial velocity, V₀

Question 26

what does this show? explain!

Answer 26

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, V₀ reaches asymptote, V_max and becomes independent of [S] = **zero-order kinetics**

Question 27

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

Answer 27

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

Question 28

what is V_max?

Answer 28

highest possible reaction rate for a given catalyzed reaction

Question 29

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

Answer 29

K_m = substrate concentration at which V₀ is 50% of V_max aka affinity of the substrate for the enzyme lower K_m = higher affinity; higher K_m = lower affinity

Question 30

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

Answer 30

purple: lower K_m so higher affinity substrate same V_max though of both substrates w/ the enzyme, even though affinities differ

Question 31

what is the michaelis-menten equation?

Answer 31

describes hyperbolic relationship between [S] and K_m of enzyme-substrate K_m is the Michaelis constant if [S] \< K_m, V₀ is directly proportional to [S] = first-order behavior if [S] \> K_m, V₀ = V_max = zero-order behavior

Question 32

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

Answer 32

slope = K_m / V_max y-intercept = 1/V_max

Question 33

how does a competitive inhibitor work? what is its apparent effect on K_m ​and on V_max?

Answer 33

competes for the same binding site as the substrate increases K_m no effect on V_max

Question 34

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

Answer 34

Question 35

how does a noncompetitive inhibitor work? what happens to Km? what happens to V_max?

Answer 35

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 V_max is reduced (higher on Y-axis) K_m is unchanged (same on X-axis)

Question 36

how does an **uncompetitive inhibitor **work? what happens to Vmax and Km?

Answer 36

binds when the substrate is bound, b/c ES complex creates a binding site for the inhibitor ## Footnote **V_max and K_m both are reduced**

Question 37

with **irreversible inhibition**, what kind of binding does inhibitor do? what happens to V_max and K_m?

Answer 37

covalent, irreversible bonding of inhibitor-enzyme decreases V_max K_m is unchanged

Question 38

what are the effects of irreversible inhibition?

Answer 38

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)

Question 39

what is an effector? what kinds are there?

Answer 39

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**

Question 40

what are homotropic and heterotropic modulation?

Answer 40

**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

Question 41

what are allosteric enzymes?

Answer 41

enzymes that're subject to regulation by effectors often are comprised of multiple subunits

Question 42

what is alpha-ALA synthase an example of?

Answer 42

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

Question 43

what is aspartate carbamoyltransferase an example of?

Answer 43

a multimeric regulatory enzyme

Question 44

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

Answer 44

homotropic modulation 1st site's occupation increases affinity of subsequent sites to bind V₀ vs [S] is **sigmoidal** (i.e. hemoglobin)

Question 45

describe model of homotropic allostery

Answer 45

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

Question 46

what is unique about glucokinase re: positive cooperativity?

Answer 46

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

Question 47

how does positive heterotropic modulation work?

Answer 47

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**

Question 48

what is heme synthesis pathway an example of? explain

Answer 48

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

Question 49

what is heterotropic effectors' impact on K_m and V_max?

Answer 49

**activators (positive effectors)**: decrease Km, no effect on Vmax **negative effectors**: increase K_m, no effect on V_max (***apparently competitive inhibition***) OR K_m unaffected, V_max reduced (***apparently non-competitive inhibition***)

Question 50

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

Answer 50

come from outside the signaling pathway therefore don't maintain homeostatic control, instead serve **signaling role **

Question 51

what is PKA activation by cAMP example of?

Answer 51

positive heterotropic modulator cAMP binding induces dissociation of catalytic and regulatory subunits of PKA therefore catalytic subunits can phosphorylate their substrates

Question 52

what is covalent modification

Answer 52

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

Question 53

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

Answer 53

phosphorylation/dephosphorylation protein kinases: add phosphate group protein phosphatases: catalyzes hydrolysis of phosphate group

Question 54

is one protein regulated by one protein kinase?

Answer 54

no, often have many phosphorylation sites on a typical protein a particular protein may have overlapping phosphorylation sites for different protein kinases

Question 55

what is a consensus sequence?

Answer 55

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

Question 56

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

Answer 56

**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

Question 57

what happens when calmodulin binds CaMKII?

Answer 57

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

Question 58

why does CamKII have a "memory"?

Answer 58

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

Question 59

what are zymogens?

Answer 59

an inactive precursor that, when proteolysed, liberates an enzyme

Question 60

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

Answer 60

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**

Question 61

how is the clotting pathway regulated? describe it

Answer 61

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

Question 62

what is an enzyme cofactor? what is its function?

Answer 62

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

Question 63

examples of important enzyme cofactors?

Answer 63

Question 64

what are **isoenzymes**? what does their existence demonstrate?

Answer 64

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

Question 65

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

Answer 65

hexokinases are 4 isozymes - hexokinase I, II, III, IV - that phosphorylate simple sugars hexokinase IV = glucokinase, is diff from the other isoforms,

Question 66

what makes glucokinase unique from the other hexokinase isoforms?

Answer 66

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 K_m for glucose** than the other hexokinases 4) it is **abundant in the liver**, not ubiquitous throughout the tissues like the other hexokinases

Question 67

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

Answer 67

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

Question 68

what is creatine kinase? where are its isozymes found, and why are its isozymes clinically significant?

Answer 68

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 **

Question 69

what is k_cat? what does it measure?

Answer 69

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

Question 70

what is the specificity constant? what does it explain? what does a large / a small value indicate?

Answer 70

K_cat/K_m 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

Question 71

what is catalytic perfection?

Answer 71

K_cat/K_m must be between 10⁸ and 10⁹ M^-1s^-1 if an enzyme's K_cat/K_m 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

Question 72

what are the 6 categories of enzymes?

Answer 72

oxidoreductases transferases hydrolasese lyases isomerases ligases

Question 73

what do oxidoreductases do

Answer 73

transfer electrons (hydride ions or H atoms)

Question 74

what do transferases do

Answer 74

group-transfer reactions

Question 75

what do hydrolases do

Answer 75

hydrolysis reactions (transfer of functional groups to water)

Question 76

what do lyases do

Answer 76

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

Question 77

what do isomerases do

Answer 77

transfer of groups within molecules to yield isomeric forms

Question 78

what do ligases do

Answer 78

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

Question 79

are all enzymes proteins?

Answer 79

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

Question 80

what's the most important/complex ribozyme?

Answer 80

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