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BioMedChem M3 > 29 - Basic Enzymology > Flashcards

29 - Basic Enzymology Flashcards

1
Q

Michaelis Menten Model

“E”+”S” ⇌ “E”∙”S” → “E”+”P”

1st step is REVERSIBLE

2nd step is irreversible
(unidirectional)

A

“E”+”S” ⇌ “E”∙”S” → “E”+”P”

Enzyme contacts substrate by diffusion to form the
E-S Intermediate

Which then passes over the activation barrier to yield:
Product = P
OR
E-S can FALL APART & the enzyme / substrate diffuse away

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2
Q

What is k+1

A
  • *BI-molecular** rate constant
  • (involves BOTH E & S, SEPERATELY)*

for FORMING E-S

L/mole-sec = molarity-1sec-1 = M-1-sec-1

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3
Q

What is k-1

A
  • *uni-molecular** rate constant
  • (starts with just ONE MOLECULE = E-S complex combined)*

for DISSOCIATION of E-S

sec-1

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4
Q

What is k+2

A
  • *uni-molecular** rate constant
  • (starts with just ONE MOLECULE = E-S complex combined)*

for BREAKDOWN of E-S into E + P

sec-1

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5
Q

What is this?

A

RATE LAW for the M-M Model

Km = Michaelis Constant

[E0] = original enzyme concentration

NO simple interpretation of MOLECULARITY or ORDER

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6
Q

What is Km

A

Michaelis Constant

it can be RELATED to a dissociation constant (Ks)
but is NOT quite the same

just the SAME UNITS

of micromolar

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7
Q

What is k+2 or kcat

A

TURNOVER Number

the MAXIMUM # of
substrate molecules converted / turned over into product

per unit time, per active site

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8
Q

What is the Maximal Rate of Reaction?

Rxn rate is maximixed when ALL of the active sites
on ALL of the enzyme molecules are Filled w/ substrate

A

Rate equation simplifies to:
Vmax = kcat [E0]

this occurs ONLY if the enzyme is SATURATED with substrate

  • generally requires that* [S] >>> Km
  • If [S] is not high enough to saturate the enzyme*, you can’t use this simple expression, but must instead use the original rate law
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9
Q

When can this rate equation NOT be used?

Vmax = kcat [E0]

A

if the [S] is NOT high enough to saturate the enzyme
we can not use that expression,

we MUST use the Original Rate law

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10
Q

Traditional Unit = U​

vs

katal = kat

A
  • *U** of enzyme activity is the
  • *amount of ENZYME** that can convert ONE micromole of substrate into products in ONE MINUTE
  • *kat** = SI unit of activity, the amount of ACTIVITY that converts
  • *ONE** mole of substrate into products in one SECOND
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11
Q

Equivilance of U to kat

A

1 microkat = 60 (traditional) Units of activity

Since:
kat=1mole vs U=1micromole

1 second vs 1 minute

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12
Q

What does this indicate? Km vs kcat

A

Km = micromolar
Takes A LOT of CO2 to saturate the enzyme

kcat = per second
there is a VERY FAST TURNOVER

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13
Q

What does this indicate? Km vs kcat

A

Km = micromolar
does NOT take a lot of substrate to saturate the enzyme

kcat = per second
there is a VERY FAST TURNOVER ​ –> produce pyruvate quickly

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14
Q

What does this indicate? Km vs kcat

elastase chews up on AA’s

A

Km = micromolar
Takes A LOT of substrate to saturate the enzyme

kcat = per second
there is a SLOW turnover

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15
Q

What does this indicate? Km vs kcat

fumarase is in the TCA cycle

A

Km = micromolar
does NOT take a lot of substrate to saturate the enzyme

kcat = per second
there is a VERY FAST TURNOVER ​

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16
Q

When can Km be APPROXIMATED by Ks for the E-S complex?

Km = Michaelis Constant

Ks = dissociation constant

A

Km =~ Ks
ONLY IF
k+2 / k+1 <<< Ks

rate of catalysis is MUCH SLOWER*** than ***rate of dissociation

_k<sub>+2</sub>_ = _rate of **breakdown** of E-S --\> E + P_ 
k<sub>+1</sub> = rate of ***dissocation*** of E + S \<-- E-S
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17
Q

What is Ks

A 
_k<sub>-1</sub> = rate of ***dissociation*** of **E + S** \<-- E-S_
k<sub>+1</sub> = rate of **formation** of E + S --\> **E-S**
  • *RATE of Dissociation**
  • only approximates Km if the rate catalysis is MUCH SLOWER than the rate at which E-S dissociates back to E + S*
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18
Q

How to determine the INITIAL RATE of an enzyme reaction?

and [Eo]

A

Set up the rxn with measured # of enzyme & substrate
then follow the progress by measuring appearance of PRODUCT

then PLOT = [Product] vs [time]

SLOPE = Rate of Rxn
(use the slope in the LINEAR REGION of the plot)

then TRACE BACK TO T=1 for [E0]

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19
Q

Initial Rate Determination

GRAPH

A

use the slope in the LINEAR REGION of the plot

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20
Q

How to find Half-Maximal Velocity = 1/2 Vmax

A

When [S] = Km , then v = 1/2 Vmax
the numerical value for [S0] = Km

First find the maximum (plateau) for the initial rate = v
then drop back in concentration of substrate = [S] to where v is
half the maximal value

then find the corresponding initial concentration of S

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21
Q

What is the Lineweaver-Burk Plot?

A

Double-Reciprocal Plot
1/v vs 1/[S]
(min/um) vs (M-1)

Km / Vmax = Slope
should be a STRAIGHT LINE
ONLY IF the enzyme obeys the Michaelis-Menten model

x-intercept @ -1 / Km
y-intercept @ 1 / Vmax

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22
Q

What type of plot is this?

A

Double Reciprocal

LINEWEAVER-BURK

only a STRAIGHT LINE slope if enzyme obeys M-M model

23
Q

Where is the X-Axis Intercept in the
Lineweaver-Burk (Double Reciprocal) Plot?

A

-1 / Km
(micromolar-1)

slope = Km / Vmax

24
Q

Where is the Y-Axis Intercept in the
​ Lineweaver-Burk (Double Reciprocal) Plot?

A

1 / Vmax

slope = Km / Vmax

(min / micromole)

25
What plot is this?
**Lineweaver-Burk**
26
What plot is this?
* *_Eadie - Hofstee Plot_** * not very useful*
27
What plot is this?
**_Hanes - Wolf Plot_** Technically superior \> lineweaver burk because there is ***_least distortion_*** to experimental ERRORS
28
When do we see a **_*Non-Linear* / CURVED Plot?_** and **WHY?**
When the **enzyme** is **NOT following that model** 2 common possibilities of curvature: may be **_MORE Kinetic STEPS_** in the mechanism that the M-M model allows **_ENZYME_** *may have* **_MULTIPLE ACTIVE SITES_** which may be **interacting** with one another **= cooperativity** (pos / neg)
29
What do we do with **CURVED PLOTS?**
**compare to "linear"** type plots to **_diagnose deviations_** from the M-M model USE **_Curve-Fitting SOFTWARE**_ + _**full non-linear rate law_** to get **accurate estimates** of the kinetic parameters **kcat** and **Km** k+2= kcat = turnover number
30
Basic ways to **measure the # of enzyme** (concentration) ## Footnote **Assays**
**_IMMUNOASSAY_** needs specific AB's, *does NOT measure **_inactive_*** enzymes **_ELECTROPHORESIS_** stain -\> scan ***_slow_*** also *does NOT measure* **_active_** *enzyme* **_STANDARDIZED RXN_** FAST, but *may call for **_coupling_*** of 2+ sucessive rxns in order to measure the product **_spectrophotometrically_**
31
**Immunoassay** **Positives & Negatives** (basic ways to measure enzyme concentration)
Need **_SPECIFIC ANTIBODIES_** to the enzyme ***does NOT measure* *_INACTIVE_* enzyme** only measures the ACTIVE enzyme think immuno**A**ssay = measures ACTIVE
32
**Electrophoresis** Positives & Negatives ​(basic ways to measure enzyme concentration)
Electrophoresis -\> stain -\> scan ***SLOW*** ***NOT for measuring*** **_ACTIVE_** **enzyme** only measures the *inactive enzyme*
33
**Stantardized Reaction** Positives & Negatives ​(basic ways to measure enzyme concentration)
Measures the ACTIVITY of enzyme **FAST** *but may call for* **_COUPLING_** of **two or more successive rxns** in order to measure the **product** **_SPECTROPHOTOMETRICALLY_**
34
How do we **monitor an enzyme** that ***does NOT*** use a **substrate or form a product?**
**_ENZYME COUPLED ASSAY_** **_UV ABSORBANCE**_ or _**FLUORESENCE_** done DIRECTLY by coupling it to a **second INDICATOR REACTION** that *itself, does NOT give a useful signal* need to add **EXCESS SUBSTRATE & Secondary ENZYME** to ensure that the **rate limiting facto**r is the **#enzyme in the first rxn**
35
**Coupled Assay Time Course** **Lag Phase -\> up to Incubation time** time **BEFORE** **products** are being made
To ensure that the: _**Overall Rate-limiting Factor** =_ **_Amount of Enzyme 1_** (1st enzyme that is or diagnostic quanitity) we need: **EXCESS** **_SUBSTRATES_** for BOTH reactions to reach SS **EXCESS** **_ENZYME_** **_#2_** the one for the indicator reaction
36
**_Aspartate Amino Transferase_** (**AST**) as an **Example of Coupled Enzyme Assay**
**AST** catalyzes this rxn: Aspartate + a-Ketoglutarate ↔ Glutamate + **Oxaloacetate** The **indicator rxn #2** uses **malate dehydrogenase** for: **Oxaloacetate** + NADH + H+ ↔ Malate + **_NAD+_** We then **follow** the **_PRODUCTION OF NAD+_** using **_FLUORESCENCE**_ or _**UV ABSORBANCE_** we need excess: reactants & 2ndary enzyme (malate dehydrogenase)
37
What type of **Inhibition** is this?
**_*Reversible* COMPETITIVE inhibition_**
38
**Competitive Inhibition**
* *inhibitor** **blocks S from binding in the active site** * various ways to block / different points of contacts* Formation of **E-S complex is *_Reduced_*,** new complex, **E-I​ is formed** *unlike* **Km, Ki**(***inhibitory constant***) is**_really a EQ dissociation constant_** (Ks) * *Competitive Inhibitor** has ***_NO EFFECT_*** on the **catalytic step** * *kcat = k+2** is THE SAME & **Vmax** is also THE SAME
39
In ***Reversible* Competitive Inhibtion** Can the **addition of MORE substrate** **OVERCOME the *inhibition?*** (restore the OG rate of rxn)
**_YES_** **apparent Michaelis constant** is **_numerically LARGER_** than **Km** so it **INCREASES** as the concentration of **INHIBITOR RISES** takes MORE substrate to HALF-SATURATE the enzyme when a competitive inhibitor is present
40
**Competitive Inhibition's** **effects on:** **kcat &** **Vmax & Km** (k+2) turnover rate / maximum velocity
**kcat & Vmax _STAY THE SAME_** **Km** ***decreases*** **_MORE substrate can OVERCOME inhibition_**
41
What type of **Inhibition Graph?**
***_Reversible_* _COMPETITIVE inhibition_** _**Increasing** **[I]** = more inhibitor leads to:_ Slope = **Steaper Slope** Y-axis (vertical) = **_Same Y-Intercept_** **Vmax** **_does NOT change_** X-axis = ***Different X-intercepts*** **Km** ***decreases***
42
What type of **Inhibition Graph?**
***_UN-_*_COMPETITIVE inhibition_** _**Increasing** **[I]** = more inhibitor concentration leads to:_ Slope = ***_No Change_*** (**Parallel Plot**) Y-axis (vertical) = **Increase Y-Intercept** **Vmax** is ***progressively reduced*** X-axis = **Increase X-intercept** **Km** is ***progressively reduced***
43
What type of **Inhibition Graph?**
**_NON-_****_COMPETITIVE inhibition_** _**Increasing** **[I]** = more inhibitor concentration leads to:_ Slope = **Increasing Slope** Y-axis (vertical) = **Increase Y-Intercept** **Vmax** is ***reduced*** X-axis = **_SAME X-Intercept_** **Km** **_does NOT change_**
44
**UN-Competitive Inhibition's** **effects on:** **kcat &** **Vmax & Km** (k+2) turnover rate / maximum velocity
*kcat idk* ***_REDUCE BOTH Vmax & Km_*** **_NOT POSSIBLE TO OVERCOME INHIBITION_**
45
**NONCompetitive Inhibition's** **effects on:** **kcat &** **Vmax & Km** (k+2) turnover rate / maximum velocity
kcat ? **Vmax _INCREASES_** **Km** **stays the SAME** **_NOT able to OVERCOME INHIBITION with more substrate_**
46
What type of **Inhibition is this?**
**_NONCompetitive / MIXED Inhibition_** **NONCompetitive = special case of mixed** inhibitor has SAME AFFINITY for either E or E-S complex **Ki = Ki'** **MIXED** Ki & Ki' are **allowed to be different**
47
What type of **Inhibition** is this?
**_UN-Competitive Inhibition_** parallel graph, vmax &km get smaller
48
**_UN-Competitive Inhibition_**
*relatively UNCOMMON*, **Parallel Plots** * *Inhibitor --\> E-S complex** * does NOT bind to the FREE ENZYME* ***_NO E-I complex is formed_*** E-S-I complex is catalytically inert still reversible **_NOT POSSIBLE to OVERCOME INHIBITION w/ MORE SUBSTRATE_**
49
**_Mixed / NON-Competitive Inhibition_**
converge to the same **X-intercept** (-1/km), same Km **Inhibitor** --\> complex with **BOTH**: **E** & **E-S** **E-I** & **E-S-I** complexes are ***_Both INACTIVE_*** ***_ADDING MORE SUBSTRATE WILL NOT OVERCOME INHIBITION_*** since inhibition can STILL OCCUR when the substrate binds
50
**alcohol dehydrogenase inhibition** **by 3-butylthiolane 1-oxide**
Example of **_UN-Competitive Inhibition_** PARALLEL PLOTS
51
**NAMPT** inhibtion by **NAD / NADH**
**_NON-Competitive Inhibition_** all **converge to same X-Intercept** (-1/Km)
52
What is the difference between **_Mixed**_ & _**Noncompetitive Inhibition?_**
**_NON-Competitive Inhibition_** a **SPECIAL CASE** of **Mixed Inhibition** where inhibitor has the **_SAME affinity_** for BOTH **E & E-S** complex *numerically,* **Ki = Ki'** * *_Mixed Inhibition_** * *Ki & Ki'** are **allowed to be DIFFERENT**
53

BioMedChem M3 (8 decks)

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