Enzymes Flashcards
State the definition and five general properties of enzymes, according to the lecturer.
- Enzymes are not altered or consumed during the reaction
- Only small amounts of enzyme are required
- Enzymes accelerate the speed at which a chemical reaction reaches equilibrium
- Each enzyme is highly specific for a given reaction
- Enzymes act by lowering the activation energy
Recognize examples of activators (inorganic cofactors) and coenzymes (organic cofactors).
a. Activators (inorganic cofactors): Zn, Fe, Cu, Mg, Mn
b. Coenzymes (organic cofactors): NAD, NADH, NADP, NADPH, Pyrixodal-5-phosphate
Explain why enzyme activity, rather than enzyme concentration, is measured in the laboratory.
The amount of enzyme is so small that it’s difficult to devise assays that are sensitive enough
The rate of product formation/amount of substrate consumed during an enzymatic reaction is measured
What is occurring during the lag, log, and substrate depletion phases
a. Lag phase – initial period when enzyme and substrate (specimen) are first mixed, but no product is formed – no change in absorbance
b. Log (linear) phase – rate of product formation is linear with time
c. Substrate depletion phase – No more substrate remaining to convert to product – no change in absorbance – rate of reaction is proportional to substrate concentration
Specifically why enzyme measurements should be made during the log phase, according to the principles of Beer’s Law
The amount of product being produced is a function of enzyme concentration
d. Definitions of zero order and first order kinetics
a. Zero order: the rate of the reaction is independent of the substrate (reactant)
b. First order: the rate of the reaction is proportional to the substrate (reactant)
- Differentiate endpoint and kinetic (multiple point/rate/continuous monitoring) enzyme methods, including why kinetic methods are preferred to endpoint methods for enzyme assays.
a. Endpoint method: the measured product is no longer changing because ALL of the analyte has reacted with the reagent in the sample – the absorbance remains fixed
b. Enzymatic Rate (Continuous Monitoring): the difference in absorbance between two (2) points during the progress of a reaction – allows for measurements to be taken while the enzyme is still reacting
Six factors that influence enzymatic activity
- Enzyme Concentration
- Substrate Concentration
- Temperature
- pH
- Presence of cofactors
- Presence of inhibitors
Recognize the importance of maintaining proper temperature and pH reaction conditions while performing enzyme assays.
The closer the assay temperature is to the normal body temp (37 C), the faster the reaction, but denaturation will occur at temperatures higher and lower than that – must be closely monitored
Buffers are incorporated into assays to maintain optimal pH and eliminate denaturation
Differentiate plasma-specific and non-plasma-specific enzymes in detail according to:
a. Definition
b. Expected relative plasma and tissue concentrations
a. Plasma-specific enzymes: known function in the plasma (i.e. coagulation factors)
Non-plasma specific enzymes: no known function in the plasma
b. Plasma-specific enzymes: higher concentration in blood relative to the tissue
Non-plasma specific enzymes: higher concentration in tissue relative to the blood
Discuss the two mechanisms for an increased rate of entry of enzymes into circulation in detail, according to:
a. Two general processes involved
b. At least two specific causes of cell damage or death
c. Two specific causes of increased enzyme production
a. Increase in the rate of enzyme release into the bloodstream
Increase in the rate of production of an enzyme
b. Hypoxia
Chemicals and drugs Physical agents (i.e. trauma)
c. Enzyme induction (via drugs, alcohol)
Proliferation of cells (that produce the enzyme -- cancer)
Recognize five physiological factors which affect enzyme reference ranges.
- Sampling time (i.e. circadian rhythm time after onset of chest pain)
- Age
- Sex
- Race
- Exercise
Discuss aspartate aminotransferase (AST) according to:
a. Three biological sources
b. Two liver diseases that gives rise to the greatest elevations
c. One muscular disease in which AST is elevated
d. One cardiac disease in which AST is elevated
e. If hemolyzed specimens are unacceptable
a. Cardiac muscle
Skeletal muscle
Liver
b. Viral hepatitis
Liver carcinoma
(Cirrhosis)
c. Muscular dystrophy
d. Myocardial Infarction: rises with 12 hrs. of onset, peaks at 18-24 hrs., normal within 4-5 days
e. YES
- Discuss alanine aminotransferase (ALT) according to:
a. Principal biological source
b. Relative use in the diagnosis of liver disease, compared to AST
c. Liver diseases in which it is elevated
d. Two liver diseases that gives rise to the greatest elevations
e. If hemolyzed specimens are unacceptable
a. Liver
b. Parallels the rise in AST activity
usually ALT > AST (persists longer as well)
AST > ALT in cirrhosis
c. Hepatic necrosis
Cirrhosis
d. Viral hepatitis
Liver carcinoma
e. YES
State the condition in which AST levels are expected to be greater than ALT levels.
Cirrhosis
Discuss alkaline phosphatase (ALP) according to:
a. Four principal biological sources of total ALP
b. Hepatobiliary diseases in which ALP is increased
c. The hepatobiliary disease which gives rise to ALP’s highest elevations
d. Bone diseases in which ALP is increased
e. Two bone diseases which give rise to ALP’s highest elevations
f. If hemolyzed specimens are unacceptable
g. Why the reference range in higher in children than in adults
a. Liver
Bone
Intestine
Placenta
b. Biliary obstruction, (Intestinal issues: GI disease & post-gastrectomy)
c. Biliary obstruction (important in the differential diagnosis of hepatic vs. obstructive jaundice)
d. Rickets, hyperthyroidism, healing fractures
e. Bone tumors and Paget’s disease
f. GROSS HEMOLYSIS not acceptable
g. Due to rapid bone growth
Discuss gamma-glutamyltransferase (GGT) according to:
a. Two principal biological sources
b. Clinical usefulness in the diagnosis and monitoring of hepatobiliary disease and chronic alcoholism
a. Liver and kidney
b. Elevated in biliary tract disorders; a marker to check for abstinence from alcohol
Contrast the relative increases of AST, ALT, ALP, and GGT in the differential diagnosis of hepatocellular vs. hepatobiliary disease.
AST and ALT are more impacted and elevated in Hepatocellular disease
ALP and GGT are more impacted and elevated in Hepatobiliary disease
All liver enzymes are affected in each disease, but these specific enzymes are greater impacted for there specific disease progression
Discuss cholinesterase (ChE) according to:
a. Three clinical applications of measuring ChE activity
b. Source?
- Assess exposure to organophosphates found in insecticides and nerve gas
- Check how patient will react to general anesthesia
- Assess presence of cirrhosis, hepatitis, liver carcinoma
b. ChE: Liver, white matter of brain, serum
Acetylcholinesterase: RBCs, CNS
- Discuss creatine kinase (CK) according to:
b. Three principal tissue sources
c. Dimeric composition and sources of its three isoenzymes
d. One muscular disease that gives rise to CK’s highest elevations
e. Cerebral diseases in which CK is elevated
b. Skeletal and cardiac muscle, brain
c. CK-BB (CK1): brain tissue, GI & GU tract
CK-MB (CK2): cardiac muscle
CK-MM (CK3): cardiac and skeletal muscle
d. Duchenne’s Muscular Dystrophy
e. CVA/Stroke
Discuss lactate dehydrogenase (LD) according to:
b. Seven principal biological sources
c. Liver diseases in which LD is elevated, including the liver disease that gives rise to the greatest elevations
d. Two types of anemias in which LD is elevated
e. If hemolyzed specimens are unacceptable
b. Blood cells, brain, kidney, liver, lung, cardiac and skeletal muscle
c. Toxic jaundice, viral hepatitis, mono, obstructive jaundice, and cirrhosis (greatest elevations in metastatic cancer)
d. Megaloblastic and pernicious anemia
e. Yes, hemolyzed specimens are unacceptable
Discuss aldolase (ALS) according to:
a. Three principal biological sources
b. One disease state in which ALS in elevated’
a. Skeletal muscle, liver, and brain
b. Duchenne’s Muscular Dystrophy
Discuss amylase (AMS) according to:
b. Two principal biological sources
c. Clinical usefulness of AMS measurements in the diagnosis of acute pancreatitis, chronic pancreatitis, and mumps
b. Pancreas and Salivary glands
c. Acute appendicitis – AMS is increased in serum, peak 24 hours, normal within 4 days
* Perforated peptic ulcer, intestinal obstruction, mumps are associated with elevated levels of enzyme*
- Discuss lipase (LPS) according to:
b. Principal biological source
c. Two diseases in which LPS is elevated
d. A comparison between the usefulness of LPS and AMS measurements in the diagnosis of pancreatitis
b. Pancreas
c. Acute & Chronic pancreatitis
d. Levels, in acute pancreatitis, parallel AMS – more specific than AMS, remains elevated 7-10 days
- Discuss glucose-6-phosphate dehydrogenase (G-6-PD) according to:
b. Biological sources
c. Disease state associated with LOW levels of G-6-PD
b. RBCs, adrenal cortex, lymph nodes, thymus, and spleen
c. Hemolytic anemia
List the four major coenzymes used in electron transfer reactions commonly employed in enzyme assays in the laboratory, including the specific wavelength used for their measurement.
NADH, NAD+, NADPH+, NADP+ – 340 nm
Definitions:
b. Cofactor
c. Holoenzyme
d. Prosthetic group
e. Apoenzyme
b. Non-protein compounds required by some enzymes to make them active
- Activator (inorganic cofactor)
- Coenzyme (organic cofactor)
c. Complete cofactor-enzyme complex
d. Bound cofactor
e. Protein portion of the enzyme
Definition:
Active site
A unique sequence of amino acids to form a groove that provides for the enzyme’s specificity for only a unique substrate
Definition
Allosteric site
A region other than the active site where a separate compound reacts, altering the shape and fit of the active site for the substrate
Isoenzyme
Different physical forms of an enzyme that catalyze the same reaction
