7) Enzymes Flashcards
3 general properties of enzymes
- not altered or consumed during rxn
- reused; only small amounts needed
- accelerate the speed of a rxn without altering equilibrium constant
heat-labile protein portion of enzyme
apoenzyme
a tightly bound coenzyme
prosthetic group
Apoenzyme and cofactor or coenzyme that form the catalytically active unit
holoenzyme
Organic or inorganic compounds that are required for enzyme function
cofactors
Organic cofactors that commonly have a structure related to vitamins
coenzymes
Enzymes that contain metal ions in their structures
metalloenzymes
4 types of enzyme specificity
- absolute specificity
- group specificity
- bond specificity
- stereospecificity
energy of activation
Amount of energy required to energize one mole of the substrate to form the activated complex.
decreased by enzymes
energy of activation
1 IU/L
Amount of enzyme that produces 1 𝜇mol of product per minute under standardized conditions of temperature, pH, substrate, and activators
3 phases of enzyme action
- lag phase
- linear phase (zero order)
- substrate depletion phase (first order)
explain zero order kinetics
Substrate is in excess (rate of enzyme reaction is independent of substrate concentration)
Rx ≠ [S]
Amount of product produced per unit of time is constant.
linear phase
zero order
explain first order kinetics
The velocity is directly proportional to the substrate concentration. Period of reduced velocity as substrate no longer in excess.
Rx = [S]
enzyme activity is measured during…
linear phase/zero order kinetics
Michaelis-Menten axes
velocity of rxn (y-axis)
substrate concentration (x-axis)
what is considered a given for each MM curve?
enzyme concentration
MM equation
V = Vmax[S]/Km + [S]
——– order kinetics occurs at Vmax
zero
Km
[S] at ½ Vmax
Vmax
When the substrate concentration is high enough that all enzyme molecules are bound to the substrate and all active sites are engaged.
—- Km = —- Enzyme affinity
↑ Km = ↓ Enzyme affinity
double reciprocal plot of MM curve
Lineweaver-Burk plot
Lineweaver-burk equation
define y, m, x, b
1/V = (Km/Vmax[S]) + (1/Vmax)
y = 1/V
m = Km/Vmax
x = 1/[S]
b = 1/Vmax
LB x-int
-1/Km
For each —– increase, the rate of reaction is doubled
10° C
Substance is similar to the normal substrate and competes with the substrate for the binding or active site of the enzyme.
competitive inhibition
When an inhibitor binds to the E S complex to form an enzyme-substrate inhibiting complex that does not yield product.
uncompetitive inhibition
Inhibitor is structurally different than the substrate and binds to an allosteric site on the enzyme molecule, which is far removed from the active site.
noncompetitive inhibition
The addition of more substrate will actually increase the inhibition
uncompetitive
Vmax unchanged
Km decreased
LB: same y-int, different slopes
competitive inhibition
Vmax decreased
Km unchanged
LB: same x-int, different slopes
noncompetitive inhibition
both Vmax and Km decreased
LB: same slopes, different intercepts
uncompetitive inhibition
Enzyme measurements are…
Product formation or substrate depletion
Multiple forms of an enzyme that can catalyze the reaction
Isoenzymes
A cytoplasmic and mitochondrial enzyme that catalyzes the reversible phosphorylation of creatinine by ATP
Creatine kinase
CK
CK important to ———— tissue
Muscle
CK found in….
Muscle
Brain
Heart
3 types of CK
CK-1, or BB—brain and CNS
CK-2, or MB—cardiac muscle
CK-3, or MM—skeletal or cardiac muscle
Elevations of C K are found primarily in conditions affecting…
brain
muscle
cardiac muscle
most common CK methodology, proceeds faster
reverse (PCr→ Cr)
3 methods of measuring CK isoenzymes
immunoinhibition, mass assay, and electrophoresis
Sandwich technique is used with two antibodies
One against the M subunit (anti-M)
One against the B subunit (anti-B)
mass assay for CK isoenzyme
Can be performed on agarose or cellulose acetate.
CK electrophoresis
C K—– migrates most rapidly toward the anode or positive pole.
C K—– migrates midway.
C K—– remains near the point of origin
BB
MB
MM
CK relative index
RI = (CKMB/total CK)(100)
RR: <3%
CK index <5%
CK index >5%
crush injury
heart attack/cardiac source
A hydrogen transfer enzyme that catalyzes the oxidation of L-lactate to pyruvate with N A D+ as a hydrogen acceptor
lactate dehydrogenase
Elevations of —- without any other information is nonspecific for any disease or disorder.
LD
In healthy individuals, the concentration of L D isoenzymes are…
LD-2 > LD-1 > LD-3 > LD-4 > LD-5
LD from heart, rbcs, and kidney
LD-1
LD-2
LD from spleen, lungs, many tissues
LD-3
LD from liver and skeletal muscle
LD-4
LD-5
MI effect on LD
LD-1 > LD-2
highest levels of LD
pernicious anemia
Most current methods measure the interconversion of NAD+ to NADH at 340 nm.
Wacker procedure
LD methodology
Wroblewski and LaDue reverse rxn
LD methodology
falsely elevates LD
hemolysis
store sample at room temp
LD sample
CK peak
CK-MB peak
24 hours
12-20 hours
tumor-associated CK
CK-BB
3 liver enzymes
Aspartate aminotransferase (AST)
Alanine aminotransferase (ALT)
Alkaline phosphatase (ALP)
Marked elevations of —– and —- are associated with hepatocellular disease or damage to hepatocytes.
AST
ALT
Marked elevation of —- is associated with hepatobiliary disease or obstructive liver disease.
ALP
Catalyzes the interconversion of amino acids and α-oxoacids by the transfer of amino groups
AST
Now generally accepted —– is of little value due to lack of tissue specificity
AST
measured using a modification of the Karmen method with the addition of coenzyme P-5-P to ensure full catalytic activity.
AST
AST
An aminotransferase that catalyzes the deamination of alanine
ALT
highest ALT elevation
acute viral hepatitis
toxic hepatitis
De Ritis Ratio (AST/ALT)
- alcoholic liver disease, cirrhosis
- Viral hepatitis, acute inflammatory disease and obstructive liver disease
- alcoholism or alcoholic hepatitis
- > 1.0
- < 1.0
- > 2.0
Wroblewski and LaDue
Uses lactate dehydrogenase (L D) as the indicator reaction.
ALT methodology
Generic name for a group of enzymes with maximum activity in p H range of 9.0 to 10.0.
ALP
frees inorganic phosphate from an organic phosphate monoester, resulting in the production of an alcohol at an optimal p H of 10.
ALP
ALP richest source
cell membranes of hepatocytes lining the sinusoidal border of the parenchymal cells and the bile canaliculi
osteoblasts in the bone
ALP elevations because of osteoblastic activity are associated with …
Paget’s disease (osteitis deformans)
normal ALP elevations
Healing bone fractures (osteoblastic activity)
Pregnancy (placental ALP)
Infants and children (growth spurts)
Diet may induce elevations in ALP activity of…
blood group B and O individuals who are secretors
Values may be 25% higher following ingestion of a high-fat meal
Bowers and McComb procedure
4-nitrophenol phosphate (4-N P P) [formerly Ρ-nitrophenylphosphate (Ρ-N P P)] is the substrate, and the yellow product, 4-nitrophenoxide, is measured at 405 nm.
ALP methodology
Transfers the γ-glutamyl group from glutathione and other γ-glutamyl peptides to amino acids or small peptides to form the γ amino acids and cysteinyl-glycine.
Gamma Glutamyl Transferase (GGT)
used to evaluate liver function, especially hepatobiliary tract disorders
Most sensitive indicator of alcoholic liver disease
GGT
used to differentiate liver disease from bone disease
GGT
not elevated in bone disease as ALP is
Hydrolyzes the phosphate group from nucleoside-5’-phosphates
5’-Nucleotidase (5’-NT, NTP)
Useful with A L P and G G T results in determining whether A L P elevation is from bone or liver disease.
5’NT/NTP
Predominantly elevated in diseases of the biliary tract where presence of bile salts stimulates its release from hepatocytes.
5’NT/NTP
Two most common substrates are:
AMP
Inosine-5’-phosphate (I N P)
5’NT methodology
2 pancreatic screening enzymes
Amylase
Lipase
A hydrolase that catalyzes the hydrolysis of complex carbohydrates including starch, amylopectin, glycogen, and their partially hydrolyzed products.
Amylase (AMY)
increased in acute pancreatitis, obstructive liver disease, acute alcoholism, ectopic pregnancy, and other conditions that affect the pancreas.
AMY
acute pancreatitis AMY peak
24 hours
non-pancreatic AMY elevation
salivary glands
especially mumps
Artifactual increase in serum AMY found in 1% to 2% of the population
Urine AMY?
Macroamylasemia
Urine AMY would be decreased (too big to pass through nephron)
Saccharogenic methods
Amyloclastic methods
Chromogenic assay
Enzymatic
Maltotetraose reaction
AMY methodologies
ACCR is elevated in …….., because the renal clearance of AMY is greater than that of CR, but returns to normal levels after the AMY is cleared from the serum.
acute pancreatitis (>8%)
Amylase Creatinine Clearance Ratio (ACCR)
ACCR = (urine AMY)(serum creatinine)/(serum AMY)(urine creatinine)
In macroamylasemia, the ACCR is…
<2%
Hydrolyzes glycerol esters of long-chain fatty acids (triglycerides) to produce alcohol and fatty acids
lipase (LPS)
LPS is less affected by ————– described under AMY, making it more specific for ————- but less sensitive.
intraabdominal conditions
acute pancreatitis
——– → Glycerol kinase → L-α-glycerophosphate kinase → Peroxidase
LPS methodology
inhibits LPS activity
hemolysis
Proteinase that hydrolyzes the peptide bonds formed by the carboxyl groups of lysine or arginine with other amino acids
trypsin (TRY)
Important in screening for cystic fibrosis and chronic pancreatitis
TRY
Serine proteinase that hydrolyzes peptide bonds connecting the hydroxyl group of tryptophan, leucine, tyrosine, or phenylalanine
chymotrypsin (CHY)
used to Investigate chronic pancreatic insufficiency
CHY
Enzyme of choice for detecting pancreatic enzymes in the feces
CHY
Group of hydrolases similar to alkaline phosphatases
Major difference is the pH of the reaction.
Acid Phosphatase (ACP)
tissue richest in ACP
prostate
Benign prostate hypertrophy (BPH) and prostate surgery.
Was also used in forensics in the investigation of rape
Bone disease is a third category of elevation
ACP
unstable at room temperature and requires immediate freezing or buffering
ACP
Catalyzes the cleavage of D-fructose-1,6-diphosphate to D-glyceraldehyde-3-phosphate (G L A P) and dihydroxyacetone phosphate (D A P)
aldolase (ALD)
Aldolase A is clinically significant for determining the primary pathology between…
muscular versus neurological myopathy
ALD main clinical significance
Diagnosing and monitoring skeletal muscle diseases
Catalyzes the hydrolysis of choline esters to form choline and the corresponding fatty acid.
neurotransmitter
Cholinesterase (CHE)
2 types of CHE
Acetylcholinesterase (true cholinesterase)
Pseudocholinesterase (acylcholine)
Pesticide poisoning
Liver function test
Abnormal genetic variants
CHE
Chronic exposure to ———- by inhalation or through the skin results in a decrease of both acetylcholinesterase and pseudocholinesterase.
organophosphates
This enzyme’s reaction is the first step in the pentose-phosphate shunt of glucose metabolism and produces NADPH
Glucose-6-Phosphate Dehydrogenase (G6PD)
clinically significant because reaction is necessary to make NADPH, required for RBC integrity
G6PD
Inherited sex-linked trait (X-chromosome)
administered antimalarial drugs or primaquine
infections
after ingestion of fava beans
G6PD deficiency
Seen in heart attacks and megaloblastic anemias
increased G6PD
