A3. Lactage dehydrogenase (LDH) catalyzes the reduction of pyruvate with NADH to

Question

A3. Lactage dehydrogenase (LDH) catalyzes the reduction of pyruvate with NADH to form lactate. A schematic of the enzyme active site is shown below, with relevant side chains from the enzyme labeled (e.g. Asp168 is the 168th amino acid of the protein sequence); the pyruvate substrate is in the center, with favorable interactions shown via blue dashes Lactate dehydrogenase In102 Arg109O NH2 NH H3C ?? 15+ Pyruvate (NADH) H CH3 ?? Ile250 Asp168 NH Arg171 (a) A mutant form of LDH in which Arg171 is replaced with Lys shows only 0.05% of the pyruvate binding of wild-type (e.g. unmutated) enzyme. Why is this dramatic effect a surprise? (b) In the crystal structure of LDH, the guanidinium group of Arg171 (e.g. the functional group containing 3 nitrogens) and the carboxylate group of pyruvate are aligned as shown above in a co-planar "forked" configuration. Based on this, explain the dramatic effect of substituting Arg171 with Lys (c) A mutant form of LDH in which lle250 is replaced with Gln shows reduced binding with NADH. Briefly explain this effect (d) Which kinetic parameter is most indicative of substrate binding effects, kcat or Km? Would you expect it to increase or decrease in the mutants described in a or b compared to wild-type?

Explanation / Answer

a) Lys has one NH2 group at side chain and one H atom which increase the effective distance to form H bond between the substrate and catalytic pocket hence the activity reduced.

b) the transition state is coplanar in Arg but in Lys, it is non-coplanar hence effective distance increases so problem in transition state formation resulting decrease catalytic activity.

c) replacement of Ile with Gly results in decrease hydrophobic interaction and overall binding of NADH to pyruvate.

d) Kcat will decrease and Km has no effect.

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