CBS - (Practical) Isozymes in Medicine Flashcards
What is Km equal to (based on Michaelis-Menten kinetics)?
Km is equal to (K-1 + K2).
If K-1 < K2, then Km is ~ K2/K1, which is not a measure of affinity.
Conversely, if K2 < K-1, then Km ~ K-1/K1, which is the dissociation constant, Kd, a measure of affinity.
Why was the spectophotometer set to read absorbance at 340 nm?
Because NADH absorbs light of wavelenght 340 nm.
Therefore, as the reactions proceeded and more NADH was created, the absorbance of the samples increased.
Why did we calibrate the spectophotometer/chart recorder system before adding the enzyme to each sample and taking the absorbance reading?
Each sample may have had a different baseline absorbance at 340 nm before we added the enzyme, so we did it to control for possible differences, as each sample started with a different lactate concentration.
LDH is an oxidoreductase that catalyses the reversible reaction:
lactate + NAD+ ⇌ pyruvate + NADH + H+
What is being reduced by LDH?
Pyruvate and NAD+ are reduced by LDH, as they both gain electrons.
Lactic acidosis can be a complication of Type II diabetes, usually due to medication prescribed to treat the condition.
The LDH isoform found in erythrocytes displays altered kinetics in Type II diabetic patients.
A Linewaver-Burk plot for the conversion of pyruvate to lactate by erythrocyte LDH from healthy controls and patients with Type II diabetes is provided.
Both groups have a similar Km, but Group B has a higher Vmax.
Which group is the healthy control, and which group is Type II diabetes patients?
Group A = healthy controls
Group B = Type II diabetic patients
We know that both groups have a similar Km, but Group B has a higher Vmax.
Therefore, erythrocyte LDH from Group B can produce lactate at a faster rate, and this is consistent with LDH contributing to the build up of excess lactate (lactic acidosis).
Why did we use initial velocity (Vi) to construct the Michaelis-Menten and Linewaver Burk plots?
Vi represent the fastest part of the reaction, before the substrate becomes consumed, or product inhibition and the back reaction occur to a significant degree,so Vi gives an accurate representation of the catalytic rate of the enzyme.
What would have happened if we had performed the assay with pyruvate and NADH (instead of lactate and NAD+) measuring absorbance at 340 nm?
The absorbance would have decreased with time because the NADH would have been consumed by the reaction.
LDH is a multi-subunit enzyme, but it displays Michaelis-Menten kinetics. Some multi-subunit enzymes are allosteric enzymes,
If Vi is plotted against [S] for these enzyme, what shape of curve is produced, and why?
A sigmoid curve, because these enzymes typically have more than one component to their reaction mechanism.
A substrate molecule binding to the first subunit increases affinity of the other subunits for the substrate, i.e leading to an acceleration of the reaction rate after a comparatively slow start. This produces a sigmoid-shaped curve.
We collected NAD+ and lactate for all the same at the same time.
Why did we add the enzyme to every sample at the same time?
All the reaction would have proceeded whilst we were measuring the absorbance changes in the first sample, and we would have missed the Vi of the other reactions and instead recorded the later, slower phase.
Assuming that K2 < K1, how will the affinity of the enzyme with a lower Km change?
It will have a higher affinity for the substrate.
An enzyme with a lower Km will require a lower concentration of substrate to reach 1/2 Vmax.
Why were the initial velocity (Vi) sections of the progress curves linear?
Because the reactions were occurring at a steady velocity, because the substrate had yet to be consumed, and neither product inhibition, not the back reaction, were occurring to a significant degree.
Oxamate alters the Vmax of LAD, but does not alter the Km.
Oxalate does not alter the Vmax of LDH, but does alter the Km.
What types of enzymes are oxamate and oxalate?
Oxamate is a non-competitive inhibitor of LDH, and oxalate is a competitive inhibitor of LDH.
