quiz 2 review ( enzymes) Flashcards
What are isoenzymes?
Groups of related enzymes that have the same active site (can catalyze the same reaction) but have different properties
What does Q10 value refer to?
Q10 = difference in enzyme activity or reaction rate when performed at 2 temperature differing by 10oC. The rate of most enzyme-catalyzed reactions will double for an increase of 10oC
What does Km refer to in an enzyme catalyzed reaction?
Michaelis constant - the substrate concentration at ½ maximum velocity (rate). It is determined to ensure that the substrate concentration used is 10 - 100 times the Km. (to ensure zero order kinetics)
What does stereochemical specificity mean and give one example.
Enzymes that only catalyze reactions of one enantiomeric form of a substrate.
i.e. either D or L form.
Example: LD will only react with L-Lactate
What is the difference between an activator and a coenzyme?
Both are cofactors. An activator is inorganic. A coenzyme is organic
What does the term specificity mean in relation to enzyme catalyzed reactions?
That enzymes will only catalyze reactions of a specific substrate (or bond) or a small number of substrates (or bonds)
What is an I.U.?
The quantity of an enzyme that will catalyze the reaction of 1 μmol of substrate per minute per L (or mL) of serum.
Written as IU/L or mIU/mL
Explain the two general approaches used to measure the extent of an enzymatic reaction
- Fixed-time (endpoint)
2. Continuous monitoring (rate or kinetic)
Describe the type of reaction that a hydrolase catalyzes
Catalyzes breakdown of substrate by the addition of H2O
Why do we monitor the activity of an enzyme catalyzed reaction during zero-order kinetics?
Because we want the reaction rate to only depend upon the enzyme concentration. Then enzyme activity is proportional to enzyme concentration
What is the principle of the BLB method for measuring ALP?
p-nitrophenylphosphate + H2O −→−Mg2+ALP p-nitrophenol + inorganic phosphate
Alkaline buffer = AMP (amino methyl propanol)
Optimum pH = 9 - 10
In the BLB method, what is the purpose of serum blanks and how are they produced?
Produced by acidifying the reaction mixture. Acid converts the p-nitrophenol from yellow to colorless. Allows a correction for the color of serum
Which type of liver disease produces the highest levels of ALP in plasma or serum?
Extrahepatic obstruction
The highest levels of ALP are seen in which type of bone disease?
Paget’s Disease (bone resorption)
If Alkaline Phosphatase (ALP) and Acid Phosphatase (ACP) catalyze the same general type of reactions, what is the main difference between the two enzymes?
Their optimal pH.
ALP - pH 9 - 10
ACP - pH 4.9 - 5.0
Why are the BLB and Roy et al. methods for acid phosphatase (ACP) not preferred methods in a continuous monitoring system?
Because their products, p-nitrophenol (BLB) and thymolphthalein (Roy et al.), were colorless at the acid pH used. So the Hillman method is used because its product (α-napthol phosphate) gives a color.
Why would a hemolyzed sample not be used for measurement of acid phosphatase (ACP)?
There is a high level of ACP in red blood cells.
What is PAP?
Prostatic Acid Phosphatase - the isoenzyme that originates from the prostate
Older methods employ tartrate as a means of differentiating prostatic ACP (PAP). What is an alternate approach?
Use the methods of either Hillman or Roy et al.
PAP hydrolyzes these substrates more readily than nonprostatic isoenzymes of ACP, so any reaction is due to PAP
Very high elevations of acid phosphatase (ACP) are seen in which clinical condition?
Metastatic prostate cancer
So it is most useful to confirm metastatic prostate cancer and to stage prostate cancer.
What is PSA and what is its clinical utility?
PSA = prostate specific antigen
Clinical use - monitoring of treatment for patients with known prostatic carcinoma. Also used in diagnosis along with rectal exam and ultrasound.
Why is human amylase called alpha-amylase?
It hydrolyzes α 1,4-glucosidic linkages of polyglucans.
Why were defined substrate methods introduced to measure amylase?
Because of problems with older starch based methods. i.e. variability of starch substrate and inconsistency of hydrolysis conditions.
How can amylase be measured by O2 consumption?
Starch −→−−−−− (Amylase) maltose, maltotriose →GlucoseGlucose + O2 −→−−−−−−−−(Gluose, Oxidase) H2O2 + gluconolactone
Decrease in O2 measured.
Why is amylase the only enzyme that is routinely measured in urine as well as serum?
Because it appears in urine due to its small size
What is the systematic name, practical name and abbreviation for the enzyme that catalyzes the following reaction?
L-lactate + NAD+ ⇄ ? Pyruvate + NADH + H+
Systematic name: L-Lactate:NADoxidoreductase
Practical name: Lactate Dehydrogenase
Abbreviation: LD
Why do most methods for aspartate aminotransferase (AST) now incorporate P-5-P in the test procedure?
Because it is a prosthetic group for AST and it increases the rate of the reaction
Why is aspartate aminotransferase (AST) used as an aid in the diagnosis of myocardial infarctions?
It begins to rise in the blood about 8 hours after AMI.
Peaks about 24 hours.
Remains elevated for about 5 days.
Can be used to confirm diagnosis of AMI.
Why is Gamma-glutamyltransferase (GGT) referred to as a transferase?
Because it transfers glutamate from one peptide to another
Highest levels of gamma-glutamyltransferase (GGT) are seen in which clinical conditions?
30 times normal elevation in intrahepatic or posthepatic biliary obstruction and in liver cancer.
Why is a wavelength of 340 nm used in NAD/NADH reactions?
Because of the difference in absorbance of NAD+ and NADH at 340 nm.
NADH absorbs at 340 nm, so an increase in absorbance can be read.
NAD+ doesn’t absorb light at 340 nm, so a decrease in absorbance can be read.
What is the basic principle for measurement of lactate dehydrogenase (LD) using a method based upon the P → L reaction? What is the optimum pH?
Pyruvate + NADH −→−− ( LD ) Lactate + NAD+
Measure decrease in absorbance at 340 nm
Optimum pH 7.1 - 7.4
a) Lactate Dehydrogenase (LD) isoenzymes are made up of how many peptide chains?
b) What kind of chains are they?
c) This gives rise to how many LD isoenzymes?
a) Four peptide chains
b) M (muscle) and H (heart) chains
c) 5 isoenzymes (LD-1 to LD-5)
What is the predominant LD isoenzyme(s) found in:
a) the heart
b) the liver
c) skeletal muscle
a) *LD-1, LD-2
b) LD-4, *LD-5
c) LD-4, LD-5`
Why will hemolysis increase serum LD values?
RBCs have a high concentration of LD
How long will serum lactate dehydrogenase (LD) levels remain elevated after AMI?
7 - 12 days post-infarct.
What is the reverse reaction for measuring serum creatine kinase (CK) and why is it the preferred method?
Phosphocreatine + ADP →Creatine + ATP
It occurs faster than the forward reaction.
Why should hemolyzed serum samples not be used for the measurement of creatine kinase (CK) activity?
Because RBCs have a high level of the enzyme AK (adenylate kinase) which catalyzes a reaction to produce ATP.
Since the CK reaction also measure ATP, it can give a falsely increased result
Why are CK isoenzymes called dimers?
They contain two subunits, M and/or B.
Di = 2
What is the predominant creatine kinase (CK) isoenzyme found in the serum of healthy individuals?
CK-MM (CK-3) is the major isoenzyme and makes up 94 - 100% of the total CK.
What can be considered one of the most specific indicators of AMI?
Increased CK-MB (>6% of total CK)
Name two instances when creatine kinase (CK) activity could be elevated in the serum of healthy individuals?
After strenuous muscular activity - up to 48 hours.
After intramuscular injections - up to 1 week.
What enzymes/isoenzymes are important in the investigation of suspected AMI?
CK (CK-MB)
LD (LD-1 & LD-2)
AST`
