lecture 3: enzymes

Question 1

what are enzymes

Answer 1

1. Enzymes are proteins or RNA molecules (ribozymes) that are powerful and specific

Question 2

what do enzymes change and keep the same in any reaction

Answer 2

They speed up the forward and reverse reactions and do not change the Keq, the free energy change of the reaction, ΔG, nor the standard free energy change, ΔGo.

Question 3

Mechanism of Enzyme Catalysis: binding

Answer 3

Enzymes contain an active site, which binds substrate, the transition state, and product with multiple noncovalent interactions. As the substrate binds, the enzyme conforms around it (induced fit).

Question 4

how does the active site of the enzyme make the change to the product more likely?

Answer 4

The active site reduces the free energy difference between the transition state and the substrate ( Ea).

Question 5

what are the ways that the enzyme, at the active site, reduces the Ea (physically speaking)?

Answer 5

a. Gathering substrates together in favorable orientation (proximity and orientation)
b. Creating a microenvironment to favor catalysis (e.g. nonpolar, acid-base, metal ion cofactor, or temporary covalent attachment)
c. Positioning of R groups such that the transition state has the greatest binding energy and is, therefore, stabilized

Question 6

cofactors

Answer 6

Some enzymes require cofactors (metal ions or coenzymes such as NAD+) to accomplish catalysis.

Question 7

holoenzyme

Answer 7

enzyme + cofactor

Question 8

prosthetic groups

Answer 8

Coenzymes that are always bound to the active site are called prosthetic groups.

Question 9

Reaction Velocity

Answer 9

product formed (or substrate lost) per time (e.g., micromoles of carbonic acid formed per minute by the action of carbonic anhydrase

Question 10

Zero order reaction velocity: substrate

Answer 10

velocity doesn’t change as [substrate] changes

Question 11

First order

Answer 11

a. velocity changes linearly with [S]

Question 12

Second order

Answer 12

velocity changes with the product of [A] and [B] or [A] x [A]

Question 13

enzyme kinetic plot- what order is it?

Answer 13

starts out first order and ends up zero order as [S] increases.

Question 14

Michaelis and Menton worked out a different formula based on the idea that an enzyme-substrate complex forms (ES) and thus:

Answer 14

the concentration of enzyme available limits the velocity when all sites are temporarily occupied by substrate (maximum velocity, Vmax).

Question 15

definition of Km

Answer 15

a measure of the affinity of the enzyme for its substrate. High Km= low affinity

Question 16

Vmax depends solely on the ___

Answer 16

enzyme

Question 17

KM is the substrate concentration at which Vo =

Answer 17

½ Vmax

Question 18

The maximum velocity, Vmax, is achieved when all enzymes are in ___

Answer 18

the enzyme-substrate form

Question 19

turnover number

Answer 19

how fast one enzyme converts substrate to product, e.g. carbonic anhydrase is 600,000 molecules per second

Question 20

The enzyme efficiency

Answer 20

kcat/Km, taking into account both speed and affinity. This parameter is used to compare which substrate the enzyme prefers or which isozyme is the most efficient at catalysis, for example

Question 21

Lineweaver-Burke

Answer 21

plot of 1/Vo and 1/[S], which yields a straight line

Question 22

Competitive Inhibition

Answer 22

the inhibitor binds to the active site and blocks the substrate

Question 23

what changes/stays same in competitive inhibition

Answer 23

Km is higher and Vmax is unchanged because high [S] swamps out inhibitor effect.

Question 24

Noncompetitive Inhibition

Answer 24

the inhibitor binds to either E or ES and blocks catalysis. ‘changes shape’

Question 25

what changes/stays same in allosteric inhibition?

Answer 25

a. Vmax is lower and Km is unchanged because active [enzyme] is lower due to [ESI] or [EI].

Question 26

Uncompetitive Inhibition

Answer 26

the inhibitor binds to ES and blocks catalysis

Question 27

what changes/stays same in uncompetitive inhibition

Answer 27

Km is lower because of LeChatlier’s principle: formation of ESI depletes ES and draws E + S to form more ES, which appears the same as higher affinity of E for S (i.e., lower Km). The percent of E removed to ESI appears that the [enzyme] is lower so Vmax is also lower.

Question 28

describe an allosteric enzyme, what it looks like and its conformations

Answer 28

All have multiple subunits (tetramers) with active sites and regulatory sites. The subunits have at least two conformations termed T for tense and R for relaxed

Question 29

T vs R states and what they mean

Answer 29

The T state has less activity and less affinity for Substrate than the R state.

Question 30

T/F: allosteric enzymes do not conform to michaelis-menten kinetics

Answer 30

True; display an s –shaped curve

Question 31

cooperative’ behavior

Answer 31

noncompetitive behavior

Question 32

why does the s-shaped curve look that way?

Answer 32

At low substrate levels, has a hard time getting started. As s increases to an intermediate level, zoomed up- velocity increases GREATLY in response to a tiny change

Question 33

S-shape of curve is due to ___ effect

Answer 33

homotropic

Question 34

homotropic effect vs heterotropic

Answer 34

Homotropic is where the molecule that induces the effect is similar to the substrate

Heterotropic is when the molecule that induces the effect is different from substrate.

Question 35

What are the two models of homotropic effectors?

Answer 35

1) Concerted - dimeric enzyme, binding of first substrate has effect on binding of second substrate
2) Sequential -four subunits, binding of 1st substrate facilitates binding of following substrates

Question 36

Inhibitors: stabilize __ states
Activators: stabilize __ state

Answer 36

T, R

Question 37

how are allosteric enzymes regulated?

Answer 37

by feedback inhibition; regulated by one or more end products of the pathway or pathways under their control

Question 38

allosteric enzymes catalyze the ___ step in a pathway

Answer 38

committed

Question 39

Positive feedback-

Answer 39

Two pathways cooperate to form a single product

Question 40

homeostasis

Answer 40

Levels of intermediates in these pathways always stay around the same

Question 41

T/F: T and R are in equilibrium

Answer 41

True

Question 42

T/F: R state is more enzymatically active than T

Answer 42

true

Question 43

concerted model

Answer 43

If a substrate binds to the 1 R one, whole thing turns into R. (all 4 units) now 4 R’s .

Question 44

Sequential model:

Answer 44

when one substrate binds, all the adjacent subunits flip shape too. But it doesn’t require that they shift AT THE SAME TIME (in concert); they can shift in a sequential manner. One by one

Question 45

the disruption of the T/R equilibrium by substrates

Answer 45

homotropic effect

Question 46

disruption of T/R equilibrium by regulators

Answer 46

heterotropic effect

Question 47

effect of regulators on the s curve?

Answer 47

shift it one way or the other - activator increases it, T state/inhibitor decreases it

Question 48

do all enzymes take on the same conformations?

Answer 48

no; MM curves are averages of all enzyme activity

Question 49

a___inhibitorcan bind whether or not the substrate is bound,

Answer 49

noncompetitive

Question 50

Uncompetitive: ONLY BINDS TO :

Answer 50

E-S complex

Question 51

When delta G is positive, the reaction does not occur spontaneously, and is:

Answer 51

endergonic

Question 52

standard Gibbs free energy of formation

Answer 52

The standard Gibbs free energy of formation of a compound is the change of Gibbs free energy that accompanies the formation of 1 mole of that substance