Enzymes

Question 1

What are cofactors?

Differentiate.

Answer 1

needed by some enzymes in order to function

• prosthetic groups: organic mol. covalently bound to an enzyme → non-dissociable
• coenzymes: organic mol. non-covalently bound to an enzyme → dissociable
• metall ions: bound to sec. interactions

Question 2

What is the native conformation of enzymes?

Answer 2

maximal catalytic activity at distinct ph and temp.

• pH: optimum is NEVER at IP
• T: ↑ T → ↑ E_kin → ↑ activity, but if T too high, denaturation

Question 3

How do enzymes change reaction kinetics?

Answer 3

change reaction mechanism
BUT: catalyze in both direction

• decreased E_a
• unchanged ΔG
• unchanged EQ-state, but decreased time to reach EQ
• unchanged thermodynamics (half life, reaction order, etc.)

_{REMEMBER diagram}

Question 4

What is enzyme activity?

Formula + unit.

Answer 4

amount of substrate converted to product per unit time

EA = -Δ[S]/Δt = Δ[P]/Δt

measured in [Catal] = [mol/sec], [μmol/min]

Question 5

What is specific enzyme activity?

Unit.

Answer 5

amount of product formed in a given amount of time per mg of total protein

→ enzyme activity per per mg of total protein

in [kat/mg]

Question 6

Define turnover number.

Formula + unit.

Answer 6

max. amount of products that is formed by a single catalytic site per second

k_cat = n_p / n_E * t

in [mol(P) / mol(E)*sec]

Question 7

What is an important medical application of enzyme activity measurements?

Answer 7

measurement of non-functional plasma enzymes

⇒ marker enzymes for blood plasma indicate died tissue cells → information abt tissue problems

ex: after heart attack

• creatine kinase CPK
• lactate dehydrogenase LD
• α-hydroxbutyrate dehydrogenase HBDH

Question 8

Explain the 2 different enzyme models.

Answer 8

enzyme-substrate complex ES stabilized by sec., ionic, covalent bonds

lock and key model:

S + E have specific shape → fit exactly into one another

induced fit model:

E changes slightly its shape as S binds

Question 9

Distinguish btw different enzyme mechanisms.

Answer 9

• catalysis by proximity: enzyme brings together substrates in proper orientiation, incr. the prob of succesfull collisions
• acid-base catalysis: enzyme transfers H⁺/OH^- from one substrate to another
• covalent catalysis: enzyme forms covalent bonds substrates, providing an energetically more favorable reaction partner

Question 10

What are proteases?

What is the most important group among proteases?

List some examples.

Answer 10

cuts proteins by hydrolysis of peptide bonds

Ser-proteases = Ser in active center

• trypsin, chymotrypsin
• elastase
• thrombin + other blood coag. factors

Question 11

What is DIPF?

Why is it pretty cool?

Answer 11

diisopropylphosphofluoridate

• *irreversible inhibitor of Ser-enzymes**
• (ex: chymotrypsin + acetylcholinesterase)*

→ used as biochemical weapon

Question 12

What are zymogens?

Answer 12

proenzymes = inactive enzyme precursors

Question 13

What are trypsinogen and chymotrypsinogen?

Explain their proteolytic activation pathway.

Answer 13

trypsinogen + chymotrypsinogen stored as zymogens to protect pancreas from autodigestion

1. activation of trypsinogen → trypsin
2. activates chymotrypsinogen → π​-chymotrypsin
3. cut each other into shorter fragments → α-chymotrypsin = active peptidase
4. trypsin inactivated by inhibitor → chymotrypsinogen activation ceases

Question 14

What is the catalytic triad in case of chymotrypsin?

What type of catalysis is it?

Answer 14

proton shuttle in active center of Ser-proteases

here: ¹⁰²Asp ⁵⁷His ¹⁹⁵Ser

→ covalent catalysis

Question 15

Explain the reaction mechanism of chymotrypsin.

Answer 15

catalysis of peptide bond

1. S binds to ¹⁹⁵Ser, shifts -OH H⁺ to ¹⁰²Asp
2. nucleophilic attack of Ser O on C of peptide bond → acyl-enzyme intermediate
3. H⁺ shuttled to peptide → H₂N-R pinches off (= 1^st product) when replaced by H₂0
4. H₂0 withdraws H⁺ to ¹⁰²Asp
5. OH^- attacks acyl-enzyme intermediate → H⁺ shuttled back to ¹⁹⁵Ser
6. original conformation restored → COOH-R pinches off (= 2^nd product)

Question 16

Explain the specificity of the Ser-proteases chymotrypsin, trypsin and elastase.

Answer 16

depends on R-side chain

• chymotrypsin: big, non-polar pocket
  → only aromatic rings: Phe, Tyr, Trp
• trypsin: negatively charged pocket
  → only pos. charged: Arg, Lys
• elastase: small pocket
  → only small -R: Gly, Ala, Ser

_{REMEMBER: chY, tYr. And just know the rest}

Question 17

What is the v_i of enzyme kinetics?

Which conditions must be fulfilled in order to measure it correctly?

Answer 17

initial reaction rate
rate (approx. linear) where highest amount of products is formed by enzyme

conditions for the measurement:

• rel. short time
• molar excess of S over E
• reverse reaction negligible

Question 18

What is the Michaelis-Menten equation?

Consequences?

Answer 18

describes enzyme kinetics
→ the lower K_M, the greater v_i

consequences:

• low substrate conc: v_i = v_max*[S]/K_M
• substrate conc = K_m: v_i = v_max/2
• high substrate conc: v_i approaches v_max
  → all E in ES complex

Question 19

What does the Michaelis constant describe?

Answer 19

• low K_M = enzyme has high affinity for substrate
• high K_M = enzyme has low affinity for substrate

Question 20

What is the Lineweaver-Burk plot?

Another name.

Answer 20

double reciprocal plot
instead of V_i vs [S], 1/V_i vs 1/[s]

• y-intercept = 1/V_max
• x-intercept = -1/K_m
• slope = K_M/V_max

_​⇒ used to determine K_M, V_max

Question 21

What are isoenzymes?

Characteristics?

Answer 21

enzymes that differ in AA sequence but catalyze the same reaction

• coded by diff. genes
• may have diff. kinetic parameters (v, K_M)
• same substrate specificity, BUT may also catalyze other substrates
• activated under diff. circumstances

Question 22

Compare the K_m of btw glucokinase and hexokinase.

Answer 22

• hexokinase: always active to supply O₂ dependent tissues w/ glucose (↓ K_M → ↑ affinity)
• glucokinase: only active when hyperglycaemia, e.g. after meal (↑ KM → ↓ affinity)

Question 23

What is lactate dehydrogenase?

Answer 23

tetrameric enzyme consisting of 2 monomer types:
H (heart) and M (muscle) → 5 LDH isozymes:

• HHHH predominates in heart tissue
• HHHM, HHMM, HMMM,
• MMMM in the liver

→ LDH levels rise in blood plasma, injured tissue type can recognized by isozyme pattern (marker enzyme)

Question 24

Differentiate btw mechanisms of irreversible inhibitors like poisons.

Examples.

Answer 24

• action on active center: covalent bond on act. center (e.g. DIPF)
• nonspecific inhibitors: denaturation (e.g. heavy metals, heat)

Question 25

What is competitive inhibition? What are its effects on enzyme kinetics?

Answer 25

**substrate analogue binds to active site** → prevents [ES]-complex formation * *V_max** unchanged _{(when S binds to E, E still functioning properly)} * *K_M ↑** → affinity ↓ _{(more molecules present, competition for S to bind)}

Question 26

How can competitive inhibition be overcome? Depends on... ?

Answer 26

_can be overcome_ by **increasing [S],** exact amount depends on: * **[I]** * **K_I (**affinity of I for E) * **K_M** (affinity of E to bind to S)

Question 27

What is non-competitive inhibition? What are its effects on enzyme kinetics?

Answer 27

bind to E (**not on active site**), but affect E's **catalysation activities** during the time of binding * *↓ V**_{max (due to decr. activity)} * *K_M unchanged** _{(no competition for S binding to act. site)}

Question 28

What is uncompetitive inhibition? What are its effects on enzyme kinetics?

Answer 28

inhibitor **binds only to ES-complex** * *V_max ↓** _{(takes longer for substrate/prod. to leave act. site)} * *K_M ↓** → affinity ↑ _{(b/c less active enzyme complexes present, less competition)}

Question 29

What is allosteric regulation?

Answer 29

**substance** binds to **allosteric binding site** and induces a **conformational change**

Question 30

What are K, V type inhibitors? How do they effect enzyme kinetics?

Answer 30

​types of allosteric inhibitors, can be activating/inactivating * _K type:_ change **K_M** * _V type:_ change **V_max**

Question 31

What is a cooperative enzyme? What are the 2 types of cooperativity?

Answer 31

**multimeric enzyme** that binds **substrate at 2+ sites** * **homotropic** cooperativity * **heterotropic** cooperativity

Question 32

What is homotropic cooperativity?

Answer 32

S binds to catalytic subunit → **activates other cat. subunits** **tense** (low affinity) → **relaxed** (high affinity) state *ex: hemoglobin when O₂ binds*

Question 33

What is heterotropic cooperativity? Example.

Answer 33

**activation or inactivation of further cat. subunits upon ligand binding changes w/ different ligands** *ex:* ATCase resp. for 1^st step of synthesis of pyrimidine is activated by ATP, inactivated by CTP