Lecture 15 - Enzyme kinetics II Flashcards
Describe the methods of determining pre-steady state kinetics.
Kcat values provide an idea of the rate constant in steady state kinetics during the slowest step of catalysis
Pre-steady state kinetics
* Stopped flow apparatus -The reactants (enzyme and substrates) are held in two separate syringes and injected and mixed in the mixing chamber. The stopping syringe is pushed out and presses the trigger which triggers data collection via detection of optical changes.
* Quench flow - For when there is no convenient optical change. The reactants are again held in two separate chambers and mixed. The reaction is stopped via the injection of quenching agent that stops the reaction. The reaction is quenched at different time points until a reaction profile is complete. It is more laborious and requires more reactants than stopped flow.
Temperature jump - Following the temperature jump a new steady state will be reached and the reaction is followed until this new equilibrium is reached.
What can be learnt from pre-steady state kinetics?
Pre-steady state kinetics allows the identification of individual rate constants within an enzyme catalysed reaction.
For enzyme-catalysed reactions with multiple reaction intermediates, the rate constants for the formation of those intermediates can be followed in real time IF:
* The catalytic cycle is slow enough
The reaction intermediates have different spectroscopic properties
Identification of the slow step in catalysis:
Example - Chymotrypsin - catalysed hydrolysis of 4-nitophenyl-acetate
The rection forming 4-nitrophenol from 4-nitrophenyl-acetate. The slow step of the reaction is the reset of the enzyme which is the rate limiting step.
Example - Alcohol dehydrogenase catalysing the reaction from ethanol and NAD+ to acetaldehyde and NADH
The rate limiting step is the release of the product from the active site
This is measured by the absorption spectra of free and enzyme bound NADH which have different absorbances
How are the isoenymes of lactate dehydrogenase adapted for their function?
Lactate Dehydrogenase (LDH):
Catalytic Mechanism:
LDH catalyzes the reversible conversion between pyruvate and NADH to NAD+ and lactate.
Important residues in the catalytic site:
Arg171: Aids in orientation.
His195: Contributes to the catalytic mechanism by aiding electron transfer.
Isoforms and Tissue Specificity:
Different isoforms are found in different tissues with varying functional specificity.
LDH is a tetrameric enzyme composed of two subunit types:
M (Muscle) and H (Heart).
Expression levels vary in different tissues.
LDH1 (Heart-Specific Isoenzyme):
Low Km for Substrates:
LDH1 has a lower Michaelis constant (Km) for substrates, indicating a higher affinity for pyruvate and NADH.
Adapted for efficient utilization of lactate and pyruvate in the heart, which has a high, constant energy demand.
Allosteric Inhibition:
Allosteric inhibition by an end product (e.g., pyruvate) helps regulate LDH1 activity.
Ensures precise control over lactate metabolism, channeling pyruvate towards mitochondrial metabolism when needed.
LDH5 (Liver/Skeletal Muscle-Specific Isoenzyme):
High Km for Substrates:
LDH5 has a higher Km for substrates, indicating a lower affinity for pyruvate and NADH compared to LDH1.
Adapted for situations where there is a high flux of pyruvate, such as in the liver and skeletal muscle during periods of high energy demand.
Absence of Allosteric Inhibition:
Not inhibited by end products like pyruvate.
Allows for continuous lactate production even when pyruvate levels are high, facilitating regeneration of NAD+ for glycolysis during intense muscle activity.
Functions in Different Tissues:
Heart:
High, constant energy demand.
Plasma membrane permeable to lactate and pyruvate.
Efficient substrate scavenging by LDH1, channeling pyruvate towards mitochondrial metabolism.
Skeletal Muscle/Liver:
Essential lactate production during muscle contraction to regenerate NAD+ for glycolysis.
High glycolytic flux results in abundant pyruvate, making high substrate affinity unnecessary for LDH5.
Liver:
Lactate required for gluconeogenesis via the Cori cycle.
Abundant substrate and no need for allosteric inhibition.
How are the isoenzymes of Glucockinase adapted for their function?
Glucokinase is an isoform of hexokinase that is found in the liver. Hexokinase isoforms I-III found as major hexokinase activities in other tissues.
* No feedback inhibition by G-6-P
* Much higher Km for glucose than HKI-III
The physiological significance of the kinetic properties of glucokinase
* After eating blood glucose concentrations rise
* Glucose is efficiently transported into hepatocytes: intracellular [glucose] in hepatocytes ~ blood [glucose]
* The liver takes on the major role of metabolising dietary carbohydrate
* The liver uses dietary carbohydrate to replenish glycogen stores. Any excess carbohydrate is converted to fat (fatty acids which are stored as triglyceride [TAG])
* In order to facilitate the steady and efficient metabolism of dietary carbohydrate the liver uses an enzyme that gets steadily more active as [glucose] rises and is not subject to feedback inhibition by product formation
Glucose-6-phosphate can be readily diverted to glycogenesis or used in glycolysis to provide precursors for fatty acid synthesis
