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Flashcards in Enzymes Deck (35)
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1

Enzymes

proteins that act as biological catalysts
decrease delta G to facilitate reaction
normally inside the cells; only in blood when cells are damaged

2

Enzyme reaction factors

time
substrate level
pH of mixture
temperature
enzyme concentration
activators/inhibitors that may be present

3

Inhibitors

competitive- binds to enzyme active site
uncompetitive- binds to ES complex
non-competitive - binds to other site & causes conformational change
reversible : can 'dilute' off
non reversible : alters active site permanently

4

Time

fixed or kinetic (series of times)
product formation different depending on the time

5

substrate

goal is to make so much substrate available so that it does not become a limiting factor & makes all the reactions a zero order reaction time

6

pH of mixture

most rx use pH of 7-8

7

temperature

optimume 37 in US; 30 in europe

8

enzyme concentration

originate from patient samples

9

activators/inhibitors

activators aid enzyme reaction to be optimum
inhibitors compete & reaction is not optimum (drugs in patient's system etc)

10

Km

substrate concentration at half of Vmax
exceeding Km will give you a zero order reaction

11

Enzyme class names

oxidoreductases - lactic dehydrogenase
transferases
hydrolases
lyases
isomerases
ligases

12

International Unit (IU)

the amount of enzyme which can convert 1 umol of substrate per minute to product

13

Systemic Unit (SI)

katal unit - amount of enzyme that catalyzes 1 mole of substrate per second

14

Isoenzymes

forms of an enzyme that are molecularly different but catalyze the same reaction
can usually be separated by electrophoresis or Ab-Ag reaction

15

Creatine Kinase

associated with ATP regeneration in muscle & transport systems
found in heart, muscle, & brain tissue
elevated CK associated with AMI, muscular dystrophy, CNS seizures etc etc etc

16

CK analyzer forward reaction

forward reaction
creatine + ATP w/ patient's CK -> CrP + ADP
ADP + PEP --(PEP phophatase) --> ATP + pyruvate
pyruvate + NADH +H --(LD)-> lactate & NAD
NAD IS READ AT 340 nm

17

CK analyzer reverse rx

CrP + ADP --(CK)--> creatine + ATP
ATP + glucose --(HK)--> G-6-P + ADP
G-6-P + NADP --(G6PD)-> bleh + NADPH
more commonly used
CK is from patient sample!
doesn't take as much activation energy for this reaction & works better on most machines

18

CK isoenzymes

M & B units in dimers - 3 isoforms
CK1 (BB)- brain tissue ; not usually found in serum
CK2 (MB) - heart muscle, increases 4-8 hr after AMI then normal after 48-72 hrs
CK3 (MM) - skeletal muscle, increases in muscle diseases

19

Aspartate Aminotransferase (AST)

transfers amino acid groups to make a new amino acid
coenzyme pyrioxal phosphate (vit B6)
found in cardiac, liver & skeletal tissue
SIGNIFICANT in viral hepatitis

20

AST analyzer methods

uses amino acid transferase reaction
substrate: L-aspartate
enzymes used: AST (patient's) & malate dehydrogenase
reads NAD @ 340 nm

21

LD analyzer method

uses pyruvate to lactate & the change in the coenzyme NAD
pyruvate + NADH + H -(LD)--> lactate + NAD
reaction read at 340 nm
enzyme found only in the CYTOPLASM of cells

22

LD isoenzymes

uses H & M subunits in tetramers
heart attack see LD1> LD2 (flipped from normal)
LD4 & LD5 increase in liver issues
LD3 -pulmonary

23

after suspected heart attack what tests are usually ordered?

general lab tests to see if lungs, kidneys & systems are normal
specific tests: CK, AST, LD, & Troponin

24

tests ordered when liver involvement suspected

AST, LD, ALT, gamma -GT, ALP

25

Alanine Transferase (ALT)

found more in the liver than other tissues
viral hepatitis: ALT may increase 5-10x
liver cirrhosis: AST> ALT (usually less than 5x)

26

ALT analyzer method

amino transferase enzyme
substrate: L-alanine
ALT: patient enzyme
2nd enzyme: LD
measure: NAD @ 340nm

27

Alkaline phosphatase (ALP)

NEEDS MG2+ as ACTIVATOR
found in most cell membranes as it is involved in the transfer of metabolites across cell membranes
ALP in serum: bone & liver

28

ALP associations

^^ ALP associated w/ liver or bone
liver biliary duct obstructions can lead to ^ ALP
bone diseases ^ ALP : ricketts, bone cancer, padgetts
hepatitis: modest ^ ALP

29

ALP method

ALP is a hydrolase enzyme
substrate:para-nitro-phenol phosphate (PNPP)
ALP: patient enyzme
turns yellow & read

30

ALP isoenzymes

5 possible isoenzymes
liver isoenzyme overlaps w/ bone isoenzyme
bone isoenzyme is not as heat stable as liver isoenzyme - heat stability isoenzyme separation technique