Exam 3 enzymes
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Plasma/serum enzyme concentration levels are useful in
diagnosis in disease or physiologic abnormalities
Isoenzyme
particular enzyme may exist in multiple forms in varying quaternary structures.
Isoenzymes are differentiated based on
Electrophoretic mobility, solubility, or resistance to inactivation.
Isoform
Enzyme is subject to post-translational modifications.
___ and ____ contribute to heterogeneity in properties and functions.
Isoenzyme and Isoforms
Proenzyme (zymogen)
secreted in inactive form (mostly digestive enzymes)
Inorganic co factors are composed of
Metal Ions as activators (Cl-, Mg+, Cu-)
Organic cofactors include
Coenzymes: NAD; ATP; ADP Prosthetic group: coenzyme bound tightly to the enzyme Holoenzyme: complete and active system Apoenzyme: (enzyme portion + coenzyme)
Michaelis and Menten Theory
Substrate readily binds to free enzyme at a low substrate concentration. Reaction rate (velocity) steadily increases when more substrate is added. With the amount of enzyme exceeding the amount of substrate.
Michaelis- menten constant
Km= [S] 1/2 max, indicates the amount of substrate needed for a particular enzymatic reaction.
In the michaelis-menten constant, if [S] is greater than Km by factor of 100
Follow zero order kinetics
If more than one substrate
Lowest Km is a natural substrate (not man made)
Lineweaver-burk plot
is a double reciprocal of Michaelis-Menten- yields a straight line. Most accurate and convenient to determine Vmax and Km. Reciprocal is taken of both substrate concentration and velocity of enzymatic reaction. Can be used to identify the type of inhibitor.
Optimal pH for enzymes
7.0-8.0, may denature an enzyme or influence in ionic state if >,<
Common activators:
Calcium, iron, magnesium, manganese, zinc, and potassium. Nonmetallic: bromide and chloride. If there is excess, it may inhibit reaction.
Coenzymes (organic)
Nucleotide phosphate and vitamins. When quantitating an enzyme that requires coenzymes, always provide excess to avoid dependency.
When measuring enzyme activity:
No inhibitors. Control variables. Use coupled reactions. Colorimetric. UV involving NAD and NADH (340 nm) Measured at zero order region.
Measurement of Enzyme Mass
Quantify enzyme concentration by mass. CK-MB. Can overestimate due to cross-reactivity with inactive enzymes, macroenzymes, zymogens or partially digested enzymes. Can also be quantified by electrophoretic techniques- isoenzymes/isoforms