In the following problem, please draw out the appropriate amino acids mentioned

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 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 Gln 102 109 NH Arg 246 Thr NH H3C-C-OH HN NH H CH H O C NH Pyruvate N-(NADH) 195 His CH3 H H H HN NH H3C-C-CH2 Ile250 168 Asp NH Arg171

Explanation / Answer

Q.No A

Ion-dipole interaction is formed between the side chain of charged Arg109 and the polar carbonyl of pyruvate. During catalysis, the charged Arg109 side chain stabilized with the polarized carbonyl transition state. In the mutant state, the binding is reduced to over a hydrogen bond and this leads to weaker substrate binding, catalytic activity and the lost of stabilization of the transition state.

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