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Clinical Chemistry II - 4378 > Introduction in enzymes > Flashcards

Introduction in enzymes Flashcards

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1
Q

Enzymes are mainly

A

Enzymes are mainly proteins that facilitate biochemical reactions

Ribozyme is an non protein enzyme= RNA splicing

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2
Q

Enyzmes are biological what

A

catalysts.

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3
Q

Systemic enzymes

A

Active throughout the body

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4
Q

Tissue specific enzymes

A

Active in a specfic area

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5
Q

Can measure the acitivity of these enzymes to

A

-Can measure the ACTIVITY of these enzymes in the blood to
ascertain whether these organs have been or are being damaged.
-Abnormal serum enzyme levels are found in various diseases and
inflammation

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6
Q

Cofactor/ coenzyme

A

1) Protein or non-protein
2) Permanent or temporary
3) Essential factor for the enzymes which
require cofactor

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7
Q

Competitive inhibition

A

Compete for enzyme active site

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8
Q

Allosteric inhibition

A

Binds to a seperate site other then the active site and changes the active site shape

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9
Q

Proenzyme

A

Zymogens- Inactive or less active precursor of enzyme
- Proteolytic modification required to be
activated

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10
Q

Zymogen examples

A

Angiotensinogen,
trypsinogen, pepsinogen,
chymotrysionogen, prolipase

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11
Q

Enzyme kinetics

A

-Enzymes speed up reactions
-Enzymes work by converting a substrate into a product via an
enzyme-substrate complex:

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12
Q

E + S–> ES=

A

+1

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13
Q

ES–> E+S=

A

-1

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14
Q

ES—> E +P=

A

+2

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15
Q

E+P–> ES

A

-2

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16
Q

Enzyme reaction

A

E+S–> ES—> E+P

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17
Q

Km= formula

A

(K-1+ K2)/ K1

Km= Dissociation/ Association

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18
Q

Vmax=

A

Is the reaction rate when the enzyme is fully saturated by substrate

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19
Q

Km=

A

Is the substrate concentration that is required to make the speed reach 1/2 Vmax

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20
Q

How does the enzyme speed up the reaction rate

A

1.) Lowering acitivation energy
2.) increasing the rate constant
3. increasing substrate specificity ( or Substrate concentration)

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21
Q

Reaction rate=

A

K[S]to the X power [S] to the Y power

Rate= [A]^x * [B]^y

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22
Q

Delta G=

A

Product energy- Reactants energy

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23
Q

negative delta G =

A

Exergonic or energy releaseing

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24
Q

Activation energy=

A

The amount of energy required to make a reaction move forward

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25
Q

Factors influencing the rate of the reaction

what concentrations

A
  • Enzyme concentration
    [Enzyme], the faster the product formation
    b/c more enzymes = more enzyme/substrate complexes

Substrate concentration
* Substrate readily binds to the enzyme at low concentrations.
* [substrate], the rate of reaction increases.
* But [substrate] too high, enzyme Saturation

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26
Q

The shape of a protein effects its

A

The shape of the protein affects its activity”
Anything that alters the conformation of the
protein/enzyme will have an impact on its activity.

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27
Q

pH effects what

A
  • Fluctuation of pH: alter “ionizable group(s)” on the enzyme affect
    the enzymes shape.
    **significant change in pH can cause the enzyme to denature.
  • Change equilibrium position [H+ involved reaction]
  • Enzymes require a pH: 7 – 8
  • Exceptions: alkaline phosphatase, Acid phosphatase, Pepsin
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28
Q

What enzymes dont require a pH of 7-8

A

Alkaline phosphatase, Acid phosphatase, and Pepsin

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29
Q

Creatine + ATP with enzymes Mg2+ and CK—>

A

P-creatine + ATP + H+

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30
Q

Temperate and inhibitors effects what

A

-Temperature increases increase in molecular collisions increase
in the rate of reaction.
**Too high temperature cause the protein denature decreasing the rate
of reaction.
* Optimal temperature is close to physiological temperature (37ºC)
4.Temperature
5.Presence of Inhibitor influences rate of reactions

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31
Q

Single (end) point assays

A
  • Incubate sample with substrate for a period of time
  • Measure the end absorbance (O.D.)
  • Calculate enzyme level by comparing to the [STD]
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32
Q

Kinetic assays

A
  • Incubate sample with substrate
  • Measure the absorbance over time at certain increments
  • An average change in absorbance (product formation) is
    used to calculate “enzyme activity”
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33
Q

IU or U

A

The amount of enzyme that produces 1μM
of product per minute under standard
conditions

34
Q

using data

A

-Calculate change in absorbance per minute
-Correct for dilution factor and serum volume
-Use molar absorptivity to convert absorbance to μM
-Δ absorbance / molar absorptivity x dilution factor
total volume / sample volume

35
Q

Muscle enzymes

A

CK and LDH

36
Q

Creatine Kinase

Size

Catalyzes what

A

(CK/CPK)

  • 82kD dimeric enzyme
  • Catalyzes reversible phosphorylation of Creatine
37
Q

Creatine kinase cofactor

Active site of enzyme

CK can be partially restored by

A
  • Mg 2+ is a important cofactor [too much Mg 2+ inhibit
    the CK ]
    ➡ CK can be partially restored by treating with
    antioxidant such as GSH & NAC
38
Q

Other inhibitors of CK

A

Mn2+ , Ca2+ , Cu2+ , iodoacetate, other SH binding
proteins

39
Q

At pH of 9.0 Alkaline the reaction is

A

favored reaction

the substrates Phosphocreatine and ADP are favored

Creatine with enzyme Mg2+ and CK and ATP —–> P-creatine + H+

40
Q

At pH of 6.8 the CK reaction is

A

Not favored

Creatine stays

41
Q

Creatine Kinase highest activity

A

Highest activity in the MUSCLE: skeletal & cardiac
(2500 U/g tissue) in skeletal (550 U/g tissue) in cardiac
cf. Also in the brain, kidneys, liver & GI tract.

42
Q

Creatine kinase Diseases

A
  • MI
  • Rhabdomyolysis
  • Muscular dystrophy
43
Q

CK isomers subunits

A

Subunits: M & B (40 kD each)
Combination of subunits:

Three CK isomers

44
Q

The three CK isomers

A

CK-MM (skeletal & cardiac) 98% in SK
CK-MB (Cardiac) 25-30 in Myocardium but CKMM is 70 % of myocardium
CK-BB (Brain)

45
Q

CK number one

A

CK-BB

46
Q

CK number two

A

CK-MB

47
Q

CK number three

A

CK-MM

48
Q

Electrophoresis of CK

A
  • electrode
    CK-MM
    CK-MB
    CK-BB
    + electrode
49
Q

Both subunits ( M and B) in CK have

A

Both Subunits (M&B) Have Lys residue @ C-terminu

50
Q

Lysine in M subunit in CK can

A

Lysine in M subunits can be hydrolyzed by “carboxypeptidase

51
Q

2 CK MM isoforms

A

2 Lys removed—> CK MM1
1 Lys removed–> CK MM2

52
Q

CKMB can undergo the

A

CK MB could undergo same modification: CK MB1 / CKMB2

53
Q

Other CK isomers

A
  • CK-mt: CK in between inner and outer mitochondrial membrane
    Other CK isomers
  • CK in macroform: macro CK
  • Up to 6% of hospitalized patients’ sera
  • Type I (CK (CK-BB) + Immunoglobulin (IgG); Type II oligomeric CK-m
54
Q

Clinical Significance of CK

what types of injury

A

Muscle Injury, Inflammation, Necrosis of SM and CM
“ALL Type of Muscular Dystrophy

55
Q

CK significance

Progressive Musular Dystrophy

A

highest in infancy and childhood
Female: 15 – 171 IU/L
Male: 46 – 180 IU/L
1.**Duchenne Sex-Linked MD (x chromosome)
* 50-100 x URL
* Asymptomatic Female Carrier; 3-6x increased CK
2.Severe Physical Crush Injury
* 200x URL

56
Q

Progressive Muscular Dystrophy

X chromosome disease

A

1.**Duchenne Sex-Linked MD (x chromosome)
* 50-100 x URL
* Asymptomatic Female Carrier; 3-6x increased CK

57
Q

Progressive muscular Dystrophy

Severe what

A

2.Severe Physical Crush Injury
* 200x URL

58
Q

Drugs can effect what and cause

A

3.Drugs (pharmacological dose) that could increase serum CK activity
* Statins, Fibrates, Antiretroviral, Ang II Receptor antagonist

Progressive muscular dystrophy

59
Q

3 days after Crush injury if the CK value is more then 5000U/L then

A

higher chance of developing Acute renal failure

60
Q

Measurement of CK

A

Creatine + ATP with enzymes Mg2+ and CK —-> Creatine monophosphate + ADP + H+

61
Q

CK reaction is based on the

A

Oliver- Rosalki method

Creatine monophosphate + ADP with CK enzyme—–> Creatine + ATP
PH of 6.8

ATP + Glucose with HK enzyme—-> Glucose-6-P + ADP

Glucose-6-P + NADP+ with G6PDH enzyme —–> NADPH + 6-phosphogluconate
HK = Hexokinase
G6PD = glucose-6-phosphate dehydrogenase

62
Q

CK sources of errors

A
  • Hemolysis,
  • Exercise

increased 10 U/L per Hb (1g/L), due to RBC AK

63
Q

CK reference range

A
  • Female: 15-171 IU/L
  • Male: 46-180 IU/L
  • CK-MB: less than 6% of total CK
64
Q

Lactate Dehydrogenase

Is what reaction

A
  • Reversible reaction * Reaction equilibrium favors the reduction of pyruvate
    to lactate @ physiological pH
  • Increased pH will favor the oxidation of lactate to
    pyruvate
65
Q

Too much Pyruvate or Lactate will inhibit

EDTA inhibits

A
  • Too much pyruvate or lactate inhibits LD activity
    Zn2+
  • EDTA inhibits LDH activity by binding Zn2
66
Q

LDH cofactors

A

Zn2+

67
Q

LDH pH of 8.8-9.8

A

Lactate + NAD—-> pyruvate + NADH

68
Q

LDH pH of 7.4-7.8

A

Pyruvate + NADH—-> lactate + NAD

69
Q

LDH MW

Composed of

A
  • MW: 134 kD
  • Composed of 4 peptide chains of two type: M (or A) & H (or B)
70
Q

LDH subunits

A
  • LD1 (HHHH; H4)
  • LD2 (HHHM; H3M1)
  • LD3 (HHMM; H2M2)
  • LD4 (HMMM;H1M3)
  • LD5 (MMMM; M4)

In order of decreasing anodal mobility in an alkaline medium

So LDH1 is closest to positive electrode and

LDH5 is closest to negative electrode

71
Q

Other LDH isomers

A
  • LD-X (LD-C): four X (or C) subunits
  • LD-6: from severely ill patients
72
Q

LDH significance

A
  • Invariably found only in the cytoplasm of the cell
  • Hepatic, Cardiac, Skeletal muscle, Hematological Disorders
    ➡Significantly increased
73
Q

LD 1 and 2

A

Heart, Kidney, RBC

74
Q

LD3

A

Lung, Spleen, Lymph node, WBC, PLT

75
Q

LD4 and 5

A

Skeletal muscle, Liver

76
Q

Quantification of total LD activity:

A

Monitor NADH at 340 nm

  • Serum is preferred sample [should be stored at “room temperature”
    cf. Plasma sample: more chance for PLT contamination
    LD4 & 5 are labile at cold temperature
  • Hemolytic sample (X)
    cf. 4000 times more LD in RBC than serum
  • EP separation for separation of LD isoenzymes
77
Q

Acute myoglobulin Infarction markers

A

Myoglobin, Troponin I &T, CK-MB,

78
Q

Congestive heart failure marker

A

BNP

79
Q

Elevated LDH

A

▫ LD not specific to cardiac tissue – found in various other tissues as well.
▫ Increase in serum level from ~12 - 24 h post infarc, peaks after ~ 48-72 h,
gradually returns to normal by ~ 7 – 14 day

80
Q

Elevated CK

A

▫ CK-MB is heart specific
▫ Rises about 4-8 h after infarct, peaks at 12-24 h, and returns to normal in 2-3
days CKMB index = CKMB activity/Total CK activity x 100
CKMB: <6% total CK

81
Q

Elevated Troponins

A
  • Troponins are proteins involved in muscle contraction.
  • TnI, TnT, TnC: found in skeletal/cardiac muscle
  • Screen for cTnI and cTnT
  • Following AMI, levels begin to rise ~3 – 6 h, reach peak levels in 14-24 h, return to
    normal in 5 – 10 days
82
Q

Elevated myoglobulin

A

▫ For early detection (leaks 1-3 h of onset) of AMI.
▫ Peak is reached 5 – 12 hours.
▫ Myoglobin is a small molecule (kidney can freely filter) and thus returns to
normal in 18 – 30 h after the AMI.
▫ Problem: NOT specific
- Present in all muscle cells so non-specific

Clinical Chemistry II - 4378 (28 decks)

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