In the following problem, please draw out the appropriate amino acids mentioned
ID: 163580 • Letter: I
Question
In the following problem, please draw out the appropriate amino acids mentioned – which amino acid is mutated, and the amino acids it is mutated to. Use these drawings to help in your explanations. Lactase dehydrogenase (LDH) catalyzes the reduction of pyruvate of the enzyme’s active sire is shown below; the pyruvate is in the center.
The reaction mechanism is similar to that of many NADH reductions. The transition state involved a stronger polarized carbonyl group of the pyruvate molecule:
a) A mutant form of LDH in which Arg109 is replaced with Gln shown only a 5% of the pyruvate binding and 0.07% of the activity of wild type enzyme. Why would this mutation cause this to happen? b) A mutant where Arg 171 is replaced with Lys shows only 0.05% of the wild-type’s level of substrate binding. Why may this be surprising? c) Looking at the structure above more closely, focusing on the enzyme’s interaction with pyruvate, why would a Lys not be able to replace the Arg and maintain the same level of binding? d) Ile250 is replaces with Gln and shows reduced binding of NADH. Why would this be? A LDH was engineered where Gln102 was mutated to Arg. This enzyme now reduced oxaloacetate into malate, but no longer reduces pyruvate. This LDH was converted to Malate Dehydrogenase. e) Sketch the new active site of this mutant LDH with oxaloacetate bound. f) Why does the mutant enzyme now use oxaloacetate instead of pyruvate? Circle this in your sketch. g) The scientists who mutated this enzyme were surprised to find that substituting a larger amino acid in the active site allowed for a larger substrate to bind. Why was it able to bind a larger substrate? (Consider of the charges of the nearby amino acids).
Lactate dehydrogenase 102 Gln 109 O NH Arg 246 Th NH H3C-C-OH HN NH CH H H O C Pyruvate NH N-(NADH) 195 His H CH2 H H HN NH H3C-C-CH2 250 168 NH Asp ArgExplanation / Answer
Question
Answer
a) A mutant form of LDH in which Arg109 is replaced with Gln shown only a 5% of the pyruvate binding and 0.07% of the activity of wild type enzyme. Why would this mutation cause this to happen?
In between the charged side chain of Arg109 and the polar carbonyl of pyruvate, the substrate is held in place with the help of hydrogen and ion-dipole interaction in case of wild type of enzyme. This charged Arg109 side chain is also involved in the stabilization of the polarized carbonyl transition state.
But in case of mutant enzyme, when the substrate is help only with hydrogen bond then substrate binding will automatically become weaker and you will not have transition state stabilized by ionic interactions, so there is a reduction in catalytic activity.
b) A mutant where Arg 171 is replaced with Lys shows only 0.05% of the wild-type’s level of substrate binding. Why may this be surprising?
As you know, both Lys and Arg are more or less of the same size and have same positive changes and also possesses similar properties. That is the reason why Arg to Lys mutation will go to have very little effect and that too the pyruvate will bind to Arg171 only through ionic interaction; this mutation will not have any adverse effect
c) Looking at the structure above more closely, focusing on the enzyme’s interaction with pyruvate, why would a Lys not be able to replace the Arg and maintain the same level of binding?
The two combined hydrogen bond and ion dipole interactions will occur when the forked arrangement will try to align the two positively charged groups of Arg residues with that of negatively charged oxygen of pyruvate. But in the presence of only Lys, you will have one combined hydrogen bond and ion-dipole interaction, thus the strength interaction will be reduced. This it will affect the precise positioning of the substrate.
d) Ile250 is replaces with Gln and shows reduced binding of NADH. Why would this be? A LDH was engineered where Gln102 was mutated to Arg. This enzyme now reduced oxaloacetate into malate, but no longer reduces pyruvate. This LDH was converted to Malate Dehydrogenase.
When you replace Gln with Ile250, definitely it will show effect on binding of NADH as the Ile250 is known to interact hydrophobically with the ring of NADH, and with the substitution of Gln, you will not have this type of interaction.
Question
Answer
a) A mutant form of LDH in which Arg109 is replaced with Gln shown only a 5% of the pyruvate binding and 0.07% of the activity of wild type enzyme. Why would this mutation cause this to happen?
In between the charged side chain of Arg109 and the polar carbonyl of pyruvate, the substrate is held in place with the help of hydrogen and ion-dipole interaction in case of wild type of enzyme. This charged Arg109 side chain is also involved in the stabilization of the polarized carbonyl transition state.
But in case of mutant enzyme, when the substrate is help only with hydrogen bond then substrate binding will automatically become weaker and you will not have transition state stabilized by ionic interactions, so there is a reduction in catalytic activity.
b) A mutant where Arg 171 is replaced with Lys shows only 0.05% of the wild-type’s level of substrate binding. Why may this be surprising?
As you know, both Lys and Arg are more or less of the same size and have same positive changes and also possesses similar properties. That is the reason why Arg to Lys mutation will go to have very little effect and that too the pyruvate will bind to Arg171 only through ionic interaction; this mutation will not have any adverse effect
c) Looking at the structure above more closely, focusing on the enzyme’s interaction with pyruvate, why would a Lys not be able to replace the Arg and maintain the same level of binding?
The two combined hydrogen bond and ion dipole interactions will occur when the forked arrangement will try to align the two positively charged groups of Arg residues with that of negatively charged oxygen of pyruvate. But in the presence of only Lys, you will have one combined hydrogen bond and ion-dipole interaction, thus the strength interaction will be reduced. This it will affect the precise positioning of the substrate.
d) Ile250 is replaces with Gln and shows reduced binding of NADH. Why would this be? A LDH was engineered where Gln102 was mutated to Arg. This enzyme now reduced oxaloacetate into malate, but no longer reduces pyruvate. This LDH was converted to Malate Dehydrogenase.
When you replace Gln with Ile250, definitely it will show effect on binding of NADH as the Ile250 is known to interact hydrophobically with the ring of NADH, and with the substitution of Gln, you will not have this type of interaction.
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