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Cell Biology and Signalling > Enzyme Kinetics > Flashcards

Enzyme Kinetics Flashcards

1
Q

What is enz kinetics?

A

study of the rate of an enzyme catalysed reaction, and how that rate varies with different substrate concentrations, amounts of inhibitors,
metal ions and cofactors as well as pH

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2
Q

How do you work out reaction rate?

A

change in [product]/change in time

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3
Q

What is the reaction rate?

A

decrease in the amount of substrate per unit time

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4
Q

How can reaction rate be measured?

A

increase in the amount of product formed per unit time

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5
Q

What happens at low [substrate]?

A

reaction rate is directly proportional to the substrate concentration (1st order kinetics).

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6
Q

What happens at high [substrate]?

A

reaction rate is independent of substrate concentration (zero order kinetics).

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7
Q

Why does rate increase with increasing [substrate] but then plateau as [substrate] continues to increase?

A

As u increase sub conc – chance of it colliding with active site increases but there’s finite no. of active sites – become sat. At highest conc poss of sub – no amount of increase in sub will produce increase in reaction rate. Line goes flat at infinite sub conc

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8
Q

What is Michaelis-Menton reaction model?

A 
k1       k2
E + S  ES --> E + P
           k-1
Where:
S = substrate
E = enzyme
ES = enzyme-substrate complex  
k1, k-1 and k2 = rate constants
K1 = association rate
K-1 = dissociation rate
K2 = rate of enz + product
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9
Q

What could said about k2?

A

may be same as other rates/could be greater – take as many mol coming in that E + S can throw at it or slow so that while ES is waiting to be broken down to E + P – chance of it dissociating back to E + S is high

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10
Q

What are 3 assumptions for Michaelis-Menton reaction model?

A

[S]&raquo_space; [E] so that the amount of substrate bound by enzyme at any one time is small (ES complex tiny in comparison)
[ES] does not change with time (steady-state); formation of ES is = to breakdown of ES (to E+ S and E+ P).
Initial velocities used, concentration of product small and back reaction of P to S can be ignored

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11
Q

What is Michaelis-Menton equation?

A 
Vo = V max[S ]/Km + [S ]
V0 = initial reaction velocity, measured as soon as enzyme and  substrate are mixed.
Vmax = maximal velocity of an enzyme catalysed reaction ie when  all the enzyme active sites are fully saturated with substrate.
Km = Michaelis constant = (k-1 + k2)/k1  
[S] = substrate concentration
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12
Q

What is Km basically?

A

Km basically is how quickly does the ES form and how quickly does it break down and the ratio of these constants gives Km

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13
Q

What is MM equation in words?

A

Initial velocity of enz catalysed reaction = max velocity x sub conc/Km + sub conc

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14
Q

What are units of Km and what does that allow you to find?

A

conc e.g. Molar – can find Km if u know what [S] at 0.5Vmax is

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15
Q

What is Km?

A

Km = [S] at which initial velocity is ½ max velocity

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16
Q

What is equation for Km?

A

Km = k2 + k-1/k1

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17
Q

What do Km values tell u about an enz?

A

how enz responds to change in conc of sub – do u need lots of sub for it to work or does it convert as soon as there’s sub?

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18
Q

What does Km of 25 for catalase mean?

A

For catalase to be working at half its max rate (Km) – need 25mM H2O2 (V. large amount). Below that rate is increasing in prop to amount of sub – suggest catalase wants to get rid of H2O2 from the cell. The more u have, faster enz will work to get rid of it.

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19
Q

What are ATP and D-glucose?

A

Co-sub but don’t have same Km and they’re needed to make glucose-6-phosphate

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20
Q

What is Kcat?

A

turnover number is equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate.

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21
Q

What does Kcat of 40m for catalase mean?

A

40m H2O2 mol per sec on single enz mol

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22
Q

How can u compare catalytic efficiency of 2 enz?

A

Kcat/Km, the specificity constant, (ratio of Kcat to Km)

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23
Q

Why is Km/Kcat alone not good method for measuring enz catalytic efficiency of 2 enz?

A

Two enzymes may have the same Kcat but very

different Km

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24
Q

What is Lineweaver-Burk plot equation?

A

1/Vo = Km/Vmax x 1/[S] + 1/Vmax

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25
How does MM equation relate to Lineweaver-Burk plot?
Rearranging the Michaelis-Menton equation to the form y = mx + c
26
What do all the bits in y = mx + c mean?
``` Y = y axis M = gradient X = x axis C = y-intercept ```
27
On a graph of LWB plot - what do all the bits mean?
``` Y = 1/Vo M = Km/Vmax X = 1/[S] C = 1/Vmax x-intercept = -1/Km ```
28
What are 2 clinical uses of enz measurements?
1. Differential diagnosis of disease by Investigating plasma levels of ‘escaped’ enzymes: eg Alanine aminotransferase, Creatine kinase 2, Lactate dehydrogenase. 2. Laboratory estimations of metabolites such as glucose in body fluids (blood, urine): eg glucose oxidase is used in Diastix (plasma) and Clinistix (urine) tests.
29
How would u use Vmax in clinical use of enz measurement?
To tell level of enz activity in particular tissue/fluid sample. Look for more Vmax which means more enz indicating enz normally present in plasma is there
30
How would u use [S] in clinical use of enz measurement?
Also to measure conc of sub through prop between amount of sun and enz – look at glucose conc in diastix + clinistix – way of measuring glucose conc
31
What is structure of lactate dehy?
composed of four monomers = tetramer – 4 polypeptide chains of 2 types: heart and muscle – diff genes code for them
32
How many isoenz of LDH are there?
5
33
What are 5 isoenz of LDH?
``` H4, H3M, H2M2, HM3, M4 ```
34
What info does appearance of creatine kinase and lactate dehydrogenase in plasma after myocardial infarction give?
Can measure Vmax (amount of enz) in plasma to indicate whether someone’s had a heart attack Can see whether the’rs change in the amount of creatine kinase 2 or lactate dehy in plasma During heart attack – tissue dying – leakage out of cells of the proteins so u can see or if there’s change in enz activity
35
How can chromatography/gel electrophoresis be used to measure LDH activity during mycardial infarction?
Different tissues have different levels of each of the lactate dehydrogenase isoenzymes During myocardial infarction, endothelial cells rupture, releasing their contents into the bloodstream. This increases the serum levels for lactate dehydrogenase isoenzymes 1 and 2 and can be used as a diagnostic tool. Can see where extra LDH activity came from by analysing mol profile of LDH to see if it had characteristic of heart (LD1) or liver disease. Can measure the enz and be able to say someone had a heart attack 24 hrs ago.
36
What are competitive inhibitors and e.g?
they block the enzyme active site which prevents sub binding/interfere with binding: Malonate inhibition of succinate dehydrogenase. Malonate competes with succinate for succinate dehy and binds to active site but malonate not met.
37
What are non-competitive inhibitors e.g.s?
interfere in some other way with the catalytic mechanism: reversible inhibition of a Mg2+-requiring enzyme by addition of chealator (EDTA); irreversible organophosphorus inhibition of cholinesterase.
38
How does non-competitive inhibitor work?
Inhibitor binds to ES complex but in diff site. Enz bids to sub and then inhibitor - get ESI complex so nothing happens to ESC Or can bind to enz in a way to prevent sub binding - get EI complex and so enz inactive
39
How do comp inihib affect enz activity?
alter the apparent Km, not the Vmax as have to go to higher sub conc to get Vmax but that means half Vmax (Km) shifted to right on MM plot. On LWB plot - the slop/gradient (Km/Vmax) increases
40
What happens if u have lots of sub but little comp inhib?
chances of sub being interfered with small - still get p
41
What happens if u have lots of comp inhib but little sub?
amount of product produced tiny as enz active sites being blocked up
42
What happens if u have sub and comp inhib but keep increasing sub conc?
can overcome inhibition – higher chance of sub binding to enz than inhibitor
43
What is the effect of a non-competitive inhibitor on enzyme activity?
Non – comp inhib essentially poising enz as it binds somewhere else so if u keep increasing sub conc – nothing happens but actual interaction of sub with enz unaffected hence Km is the same. So if Vmax is red but Km is same that’s characteristic of non-comp inhib
44
Describe control of angiotensin production
1. When there’s red kidney function - Reduced glomerular filtration ---> Reduced [Na] in distal tubule ---> Renin release 2. Renin catalyses cleavage of angiotensinogen protein to ang 1 by cutting polypeptide between 2 leu to release smaller peptide 3. Angiotensin converting enz cuts 2 more aa and makes mol active - get angiotensin II 4. Angiotensin II causes Peripheral vasoconstriction - To respond to increase in blood vol and Aldosterone secretion - Increase water retention – prevent dehydration
45
Describe treatment of heart failure with ACE inhibitors
Normally: Heart disease ---> dec Tissue perfusion ---> dec Renal blood flow ---> Renin release ---> Formation of angiotensin II ---> peripheral vasoconstriction ---> inc After load on heart /Release of aldosterone ---> Na+ /water retention ---> Oedema as heart not moving blood due to the perfusion ACE inhibitors (Capotril) Breaks cycle and red production of angiotensinogen – red after load on heart
46
What is acetylcholinesterase?
breaks down ACh found in neuromuscular junction (muscle contraction) and in CNS in brain (for attention and cognition)
47
Explain process of reversible inhib of AChE-I
Neostigmine sim structure to ACh - sits in 2 active sites - cut at C=O Carbamyl transferred to serine-OH Carbamyl serine slowly hydrolysed
48
How does rev inhib of AChE-I affect enz and how can u use this info in drug production?
COVALENTLY MODIFYING ENZ BUT NOT PERMANENT AS PARTICULAR PART OF MOL SLOWLY HYDROLYSED But can create drug where its not slowly hydrolysed and can control whether inhib more/less permanent
49
Describe process of irrev inhib of AChE-I
Organophosphates e.g. dyflos = high affinity for serine residues – bind to serine and attraction so strong – only way to get over it is make new enz Get phosphorylated enz and no spontaneous hydrolysis Pralidoxime causes reactivation of enz - phosphate transfered to NOH and enz reactivated
50
How does irrev inhib of AChE-I info be used in drug production?
Dev compounds which will bind to active site to COO- part, sit alongside organophosphate and become more attractive to it than serine hydroxyl group so they’ll bind to that and take it off
51
Describe 3 ways of regulating enz activity? | What are )?
1. allosteric binding sites (+/- effectors) - Mol bind to enz – change its conformation and make it more/less active Covalent modification by other enzymes. 2.Phosphorylation (kinases) or dephosphorylation (phosphatases). 3. Induction (make) or repression (red production) of enzyme synthesis.
52
What curve to enz following MM kinetics show?
Hyperbolic curve
53
What curve do allosteric enz show?
Sigmoid curve
54
What does allosteric regulation curve show?
At low conc – relationship between sub and reaction rate quite slow but point where reaction rate increases linearly
55
Describe allosteric regulation with +ve ihib
Activator binds and opens up active site so sub bind more effectively
56
Describe allosteric regulation with -ve ihib
Allosteric inhib binds at diff site and closes down active site so sub can’t bind as well
57
What is an Eg of negative allosteric effectors (inhibitors?
ATP and citrate on phosphofructokinse which inhibit it.
58
What is Eg of positive allosteric effectors?
phosphoenolpyruvate (PEP) and fructose 1,6 bis phosphate on pyruvate kinase.
59
Which aa have addition or removal of phosphate from it?
Ser, Thr, Tyr, His residues.
60
What's the effect of phos on glycogen phosphorylase?
Phosphorylation increases activity of glycogen phosphorylase (degrades glycogen).
61
Which enz catalyses Phosphorylation of serine 14 (requires ATP)?
phosphorylase kinase
62
Which enz catalyses dephosphorylation of serine 14?
phosphorylase phosphatase.
63
How does phos affect glycogen synthase?
Phosphorylation decreases activity of glycogen synthase (synthesises glycogen). Phosphorylation at several serine residues (by glycogen synthase kinase 3) inactivates the enzyme.
64
Which enz catalyses dephos of glycogen synthase?
Dephosphorylation by a phosphoprotein phosphatase.
65
What does phos/dephos of glycogen synthase allow?
coordinate regulation of synthesis and breakdown of glycogen via adrenaline and glucagon)
66
What does allosteric regulation of PFK and PK allow?
coord reg of glycolysis pathway to match demands for energy. If u have lots of mono-phosphates – need more energy – get more glucose through. Fructose 1,6-bisphosphate says to further down pathway – more C skeletons coming through – activity of enz needs to be increased.
67
If u want to convert glycogen to glucose what mech need to take place?
more glucose breakdown – shut off syn and increase degradation.
68
If u have too much glucose and u want to store it what mech need to take place?
increase syn and shut down gly breakdown done by single signal where 1 activates and 1 removed.
69
Which enz adds phosphate during protein phos and so switches it on?
Protein kinase
70
Which enz removes phosphate during protein phos and so switches it off?
protein phosphatase
71
What does change does protein phos cause?
Change may be increased or decreased activity or even catalysis of a different reaction.
72
What is Adenylylation?
Tyr residues (ATP to PPi)
73
What is Uridylylation?
Tyr residues (UTP to PPi)
74
What is ADP-ribosylation?
Arg, Gln, Cys residues (NAD to nicotinamide)
75
What is Methylation?
Glu residues (S-adenosyl-methionine to S-adenosyl-homocysteine)
76
What do high blood glucose levels lead to?
increase in insulin production. Insulin increases rate of | synthesis of key enzymes involved in glucose metabolism
77
if u have lots of glucose in diet – which enz do u want to increase by single signal /what else can u do?
Glucokinase Phosphofructokinase Pyruvate kinase Or can be red by red insulin production
78
If regulator event is sub availiabilty - what is typical effector, change and time required for change?
Effector: substrate Change: velocity Time required: Immediate What changes velocity/rate most is quickly is amount of sub (unless at Vmax)
79
If regulator event is product inhibition - what is typical effector, change and time required for change?
Effector: product Change: Vmax/Km Time required: Immediate Can use product to inhibit enz – if u don’t want too much product can allosterically/competitively inhibit it
80
If regulator event is allosteric control - what is typical effector, change and time required for change?
Effector: end product Change: Vmax/Km Time required: Immediate
81
If regulator event is Covalent modification - what is typical effector, change and time required for change?
Effector: Another enzyme Change: Vmax/Km Time required: Immediate to minutes
82
If regulator event is Synthesis or degradation - what is typical effector, change and time required for change?
Effector: Hormone or metabolite Change: amount of enzyme Time required: Hours to days
83
Out of all responses to changes in env which one does cell do immediately?
changing sub conc

Cell Biology and Signalling (7 decks)

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