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Molecular Enzymes > Mechanisms > Flashcards

Mechanisms Flashcards

1
Q

How do biological catalyts differ from ordinary chemical catalysts

A

higher reaction rates
milder reaction conditions
greater reaction specificity
capacity for regulation

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2
Q

Overview of a structure and function of a biological catalyst

A

most are proteins, although ribozymes exist

may require a co-factor ( such as inorganic ions, organic or metalloorganic molecules (coenzymes)

can be extremely specific for a substrate, and may even be stereospecific

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3
Q

Cofactors

A

Any factor required for enzyme activity or protein function.

Cofactor = inorganic ions

Coenzyme = vitamins, or a protein, can be further divided into cosubstrates and prosthetic groups

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4
Q

prosthetic group

A

a tightly or covalently associated cofactor

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5
Q

Apoenzyme

A

protien part of enzyme

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6
Q

Holoenzyme

A

complete catalytic entity, including both the apoenzyme and the non-protien constituents

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7
Q

Substrate specificity

A

active site is geometrically and electonically complemntary to the substrate

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8
Q

permissive enzymes and theri substrates

A

example = chemotrypsin, a protease that can clease hydrophobic peptides that he N-terminus, however it may also clease esters

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9
Q

How do enzymes work tho

A

provide an envrionment within whihc a given reaction is energetically more favourable

Reactive site: pocket called “active site”

  • where substrate is bound
  • form enzymes-substrate complex
  • plays a role in catalytic process
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10
Q

What do enzymes affect about a given reaction

A

They affect the reaction rate, NOT the position of the equilibrium

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11
Q

Collision therory of reaction rates

A

1 The particles must collide in order to react
2 Not all collisions result in a reaction, and the reactants must collide in the correct orientation.
- The reactants must possess a minimum energy, the activation energy to initiate the chemical reaction

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12
Q

Energy diagrams

A

Only colliding particles that are properly orientated can deliver kinetic energy into potential energy as least as large as delta G double dagger to be able to climb over the “hill” and produce products

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13
Q

Distribution of energies for ideal gases

A

note: only a small fraction of molecules have sufficient energy to react (overcome delta G double dagger)

also, at higher temperatures, more molecules possess the kinetic energy to overcome delta G double dagger, and the reaction rate increases

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14
Q

Effect of temperature on reaction rate

A

at approximately RT, a 10C increase in temperature increases a typical reaction rate by about 2-3 times

Rate =K[A]^n

where, k is temperature depended: K is only a constant if T= constant

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15
Q

Which order of reaction are most enzymes

A

first order

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16
Q

What is the purpose of the arrhenius equation

A

relates temperature to reaction rate and activation energy,

higher K = faster rate

HIgher delta G double dagger the lower the rate, and the higher the temp the faster the rate

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17
Q

What is the effect does delta G have on rate

A

delta G has no effect on rate

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18
Q

What is reaction rate linked to

A

delta G double dagger

Note: enzymes cannot force an unfavorable reaction to go in the forward direction, ie// enzymes do not affect the position of equilibrium

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19
Q

What determines the position of equilibrium

A

delta G Prime at stp,

thus the equilibrium constant is related to delta G prime STP for the overall reaction and not to the activation energy barrier delta G double dagger

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20
Q

How do enzymes solve the problem of the fact that particles need to collide in order to react

A

particles must collide in order to react, thus enzymes need to increase the frequency of the collisions

increasing the temperature, and concentration is not realistic for biological systems, however you can increase the local concentration in the enzyme active site.

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21
Q

How do enzymes solve the problem of the fact that reactants must collide i the correct orientation

A

enzymes can help to orient the reactants properly in the active site

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22
Q

How do enzymes solve the problem of the reactants needing to posses a minimum energy to overcome delta G double dagger

A

enzymes need to increase the number of reactants with the energy to overcome the delta G double dagger

the could increase the kinetic energy of the reactants (increase in temperature), but this is not realistic for biological sytems.

They can however lower the energy barrier for delta G double dagger

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23
Q

Catalytic stratagies commnly used by enzymes

A

procimity and orientation effects

preferential binding of the transition state complex

  • binding energy
  • induced fit

acid/base catalysis

covalent catalysis

metal ion catalysis

electrostatic catalysis

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24
Q

Prosimity effects

A

intramolecular reactions greatly increase the rate

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25
Q

the Fischer hypothesis

A

the Fischer hypothesis = lock and key

can explain specificity but not the efficiency

also to be noted, super tight binding can make it harder to stabilize the transition state if the substrate is bound too tightly

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26
Q

What is the purpose of favourable reactions preferentially with the transition state

A

special interactions with the transition state that are not seen with the substrate help to lower the activation energy of the transition state

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27
Q

What would happen if the enzyme only bound tightly to the substrate and not preferentially to the transition state

A

you would see a lower rate because if the enzyme bound specifically the substrate the reaction would be unfavourable, and moreover, it would also require energy to unbind the substrate.

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28
Q

What does the oxyanion hole stabalize in a serine protease

A

sp3 tetrahedral transition state

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29
Q

Why is energy required to bind a given substrate

A

enegy is required to de-solvate a substrate

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30
Q

Interactions involved in the desolvation of a substrate

A

in aqueous solution, substrates are associated with water molecules, this is also known as a hydration shell

must give this up when the substrate is bound to the enzyme active site

weaak interactions between the solvent and substrate are replaced with weak interactions between the enzyme and the substrate
-> will contribute to binding energy and facilitate catalysis

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31
Q

Delta G^R binding energy

A

refers to the favourable interactions between E and S upon forming the ES complex = “Michaelis Complex, before the formation of the TS

contributes to specificity as well as catalysis

release fo energy upon formation of ES complex might be released as head ( a local increase in temperature or KE to help achieve TS) or might be used to fuel conformational change accompanying the induced fit

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32
Q

Delta G^T binding energy

A

refers to interactions between enzyme and TS to stabilize TS and thus reduce delta G double dagger

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33
Q

Which type of binding energy is “energy saved” due to favouable interactions between E ad TS.

A

T bind

Since energy to achieve TS in the uncatlyzed reaction was never expended, there is nothing to release

energy saved

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34
Q

Whihc type of binding energy is “energy released” upond binding of S to E, due to favourable interactions

A

R bind

S had to give up favourable solvent interactions before this

driver reaction

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35
Q

What are the assumptions of the Kochland induced fit hypothesis

A

interaction is optimum between the enzyme and the TS (not the substrate)

Upon substrate binding to enzyme, the enzyme changes conformation in order to optimize the position of their reacting groups in order to favour catalysis

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36
Q

Applications of our understanding of “preferential binding of TS complex”

A

allows understanding and design of inhibitors
-> transition state analysis (TSA) are more potent inhibitors than substrate analogs

allows understanding/ design of enzymes
-> catalytic antibodies

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37
Q

Antibodies

A

the antibody will bind to the antigen via the Fab variable region, tagging the antigen, which will either impeded the activity of the pathogen or provoke th eimmune system to destroy the pathogen

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38
Q

catalytic antibody

A

normally antibodies have no catalytic activity
-> they do however bind to substrate, if it is a TSA is could in theory catalyze a reaction

Thus, a catalytic antibody is an antibody that has catalytic activity since it is raised against a TSA

  • > demonstrates the importance of this mechanism of enzyme catalysis (preferential binding of the TS)
  • > could assist int he design of new enzymes
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39
Q

Types of acid base catalysis

A

General acid cat

Gernaral base cat

concerted general acid-base cat

40
Q

General acid catalysis

A

process in which partial proton transfer from a bronsted acid (a species that can donate a proton) lowers the free energy of a reactions TS

41
Q

General base catalysis

A

process in which reaction rate is increased by partial proton abstraction by a bronsted base (a proton acceptor)

42
Q

Concerted general acid-base catalysis

A

process in whihc reaction is simultaneously catalyzed by both processes

43
Q

What kind of nucelophile is water

A

A bad one lol

44
Q

Are oxyanions good leaving groups

A

no, they are terrible

45
Q

Covalent catalysis

A

involves rate acceleration thorugh transient formation of a catalyst-substrate covalent bond

may provide an alternate, lower energy, path towards the formation of products

gers decrease in entropy to favour enyme catalysis; help in proper alignment of substrate in active site

46
Q

What is the benifit of covlalent catalysis for the reaction of acetoacetate to acetone through a schiff bace intermediate

A

multiple resonace structutres and the imine acts as an electron sink

47
Q

What are the three stages of covalent catalysis

A

1 nucelophilic reaction between catalyst and substrate to form covalent bond

2 withdrawal of electrons from the reaction centre by the now electrophilic catalyst

3 elimination fo the catalyst, though essentially the reverse of stage 1

48
Q

Good nucleophiles

A

hydroxyl group
sulfhydryl group
amino group
imidazole group

49
Q

good electrophiles

A

protons
metal ions
carbonyl carbon atoms
cationic imine (ie schiff base)

50
Q

Metal ion catalysis

A

metalloenzymes = contain tightly bound metal ions (usually transition metals)

Metal-sctivated enzymes = loosely bind metal ions from solution (usually alkali and alkaline earth metal ions)

51
Q

How to metals participate in enzyme mediated reactions

A

by mediating redox reactions through reversisble changes in the metal ions oxidation state

by binding to substrates to help orient them properly for reaction

*By electrostatically stabilizing or shielding negative charges

52
Q

Give an example of a metal electrostatically shielding negative charges in an enzyme mediated reaction

A

formation fo Mg2+ complex in hexokinase

partially shields the negative charges and influences the conformation of the phosphate groups in nucleotides such as ATP and ADP

53
Q

metal electrostatically shielding negative charges in an enzyme mediated reaction as compared to protons

A

better, metal ions can be present at high concentrations at neutral or basic pH

Metal ionss can have charges > +1

54
Q

Metal ions promote nucleophilic catalysis via water ionization

A

a metal ion’s charge makes its bound water molecules more acidic than free water, and can therefore serve as a source of -OH ions even below neutral pHs

example = human carbonic anhydrases

55
Q

How does human carbonic anhydrase solve the problem of using water as a nulceophile

A

complex water to a metal ion such that hydroxide is used as a nucleophile

metal = Zn 2+ is chelated to histidines (3x) at the active site

this in turn increases the acidity of water (@ pH of 7)

56
Q

What is the role of His 64 in human carbonic anhydrase

A

abstracts a proton from the zinc-bound water molecule. It is too far away to do it directly, however, two water molecules provide the proton via a proton shuttle

acts as a general base

57
Q

What are the three classes of serine proteases

A

cysteine proteases

aspartyl proteases

metaloproteases

all three of these solve the same problem, but in different ways

58
Q

Serine Proteases

A

Digestive enzymes

  • chemotrypsin
  • trypsin, both produced in the pancreas and released in the intestine

elastase, helps with tissue elasticity

enzymes of blood clotting cascades (serine protease)
and other closely related enzymes = serine esterases = acetylcholinesterase which cleases esteras and amide bonds??

59
Q

Challanges faceing the serine proteases (and all enzymes for that matter)

A

proximity and orientation issues

recognition of substrate/ reactants

catalysis ( increasing the rate of reaction)

product release (product blocks active site, need to release quickly)

Must be responsive tot he needs of the cell/ tissue/ organisms ie// regulation

60
Q

Which terminus do the serine proteases cleave at

A

C terminus

61
Q

Which substrate does chymotrypsin prefer

A

bulky, hydrophobic amino acids

phy, trp, tyr
R = ser. neutral

62
Q

Which substrates does trypsin prefer

A

positively charges amino acids

arg, lys

R = Asp - , binds positive groups

63
Q

Which substrates does elastase prefer

A

small neutrall amino acids

ala

2x gly replaced with val 226 and the 216 which makes a super short and hydrophobic pocket

64
Q

How the the serine proteases solve the substrate specificity problem

A

the specificity pocket

assists in substrate recognition

also helps to address the issue of proximity and orientation

65
Q

Which challanges do the serine proteases face in terms of mechanisms of reaction

A

water is a bad nu

there is an unstable oxyanion int

NHR is a bad leaving group

these all contribute to the reason why proteins don’t spontationously degrade in water

66
Q

How do the serine proteases solve the mechanistic problem

A

Thy catalytic triad, active site ser is involved in catalysis and hydrolysis

this improves the nucleophilicity, stabilizes the transition state via an oxyanion hole and also improves the quality of the leaving group

the specificity pocket is separate from the catalytic triad and helps with specificity and orientation

67
Q

Role of Asp 102 in the catalytic triad of serine protease

A

improves the nucleophilicity of His57 via electrostatic interactions

68
Q

Role of His 57 in the catalytic triad of serine protease

A

improvement of nucerophilicity of Ser195, its own nucleophilicity is improved by electrostatic interactions by Asp197

General base catalysis

in the next phase it acts as a general acid to improve the leaving group quality of Ser 195, b/c now R’-NH2 is a better leaving group

69
Q

What is the role of Ser 195 in the catalytic triad of serine protease

A

Acid catalysic of the cleavage of a peptide bond. Acid-base cat via His 57.

ser 195 acts as a nucleophile, his 57 improves its nucleophilicity

also plays a huge role because it covalently attaches the substrate to the enzyme. (intramolecular reactions are faster)

also, through sn2 type rxn drives the transition state into the oxyanion hole

70
Q

What happens is one member of the catalytic triad is removed / mutated

A

huge drop in rate, just as bad as all three mutated, removed

71
Q

Why is the general rate of reaction higher in the fully mutated catalytic triad as compared to the uncatalyzed reaction

A

oxyanion hole stabalizes the transition state, and water could still diffuse in to act as a nucleophile1

72
Q

What is the pursose of a huge enzyme given that a 2o amino acid loop is just as catalytically effective as a serine protease

A

might be specific, but cannot be regulated to suit the needs of the cell// tissue // organism

73
Q

Why are enzymes so big?

A

to allow for proper formation of a catalytic site, and for the induced fit

also yenno regulation and allosteric control

74
Q

Ketosteroid Isomerase (KSI), and site directed mutagenisis of R-Group H-bonds

A

Ki
wild type > pond mutant > conservative oxyanion mutant

in an artificial hydrophobic environment showed that a mutation of one of the R-groups that stabilized the TS decreased the rate by a log factor of 5-6

75
Q

DIPF

A

DIPF-enz kills enz = serine protease

this is an irreversible inhibitor with covalent modification

mechanism based inhibitor = suicide substrate

thus, this is a good detector of a serine protease

76
Q

Acetylcholineesterases

A

nerve gases, insectacides venoms and poisons, could also be used to treat alzheimers disease

can be irreversible inhibitors, many used therapeutically are indeed irreversible inhibitors

77
Q

Competitive inhibitor

A

diffuses into active site and can leave

78
Q

Suicide inhibitor

A

binds and mechanistically kills the enzyme

79
Q

Concepts behind enzyme desgin

A

in theory, anything that stabilizes the TS could catalize a reaction

antibodies bind tightly to their antigens (= forms extensive noncovalnent interactons)

so, if an antibody could be designed that would bind a TS, it should stabilize the TS and catalyze the reaction

however TS are too unstable, so we could use a TSA to see if it would bind

80
Q

what kind of nucleophile is CH2OH

A

hard

81
Q

what kind of nucleophil eis CH2SH

A

soft

82
Q

names of cysteine proteases

A

papin
caspase-1
Hepatitis C virus peptidase 2
cathepsin K

83
Q

What type of active site to cysteine proteases utalize

A

catalytic diade

84
Q

Carboxypeptidases

A

Enzymes that hydrolyze peptide bonds at the C-terminus end of a protein or a peptide

includes serine, cysteine, and metallo-carboxypeptidases

85
Q

Carboxypeptidase A

A

A for aromatic

prefer to cleave peptid ebonds of C term with side cahins that contain aromatic or branched hydrocarbon chains

86
Q

Carboxypeptidase B

A

B for basic
prefer to cleave peptide bonds of C term with side chains that contain positively charged amino acids

ex lysine and arginine

87
Q

Panceratic exopeptidases: CPA1 and CPA2

A

A carboxypeptidase A
critical for may processes in the body including digestion, post-translational modification of proteins, blood clotting and reproduction

88
Q

Active site of carbozypepidase A

A

single polpeptide with active site zinc

Zn2+ coordinated with two His and one Glu

89
Q

Conformational change of Carboxypepidase A (and other enzymes) is important for

A

Induced fit

Tyr 248 moves and swings when the substrate binds

closes active site cavity

completes conversion from water-filled region into a hydrophobic one

90
Q

Induced fit

A

catalytic groups of the enzyme are brought into the correct orientation by the binding of substrate

91
Q

Kinases and induced fit

A

hexokinase
Kinases catalyze the transfer of gamma-phosphoryl of ATP to R-OH

Problem= need to prevent transfer to water

would be devastating to catalyze simple hydrolysis of ATP instead of a phosphoryl transfer

problem solved with an induced fit

92
Q

examples of Aspartic proteases

A 
pepsin
chymosin
Cathepsins
Renin
beta secretase
HIV-1 protease
93
Q

Pepsin

A

digestive enzyme found in the stomach

broad specificity: will highest 20% of ingested amide bonds
cleaves preferentially on N terminus of hydrophobic preferably aromatic AA residues

94
Q

How are we attempting to treat HIV

A

aspartyl protease

95
Q

HIV protease structure

A

is a dimer, with a single active site formed by two identical symmetrically arranged subunits

active site aspartic acid residue

flaps close down after substrate bound

Molecular Enzymes (7 decks)

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