The structures of the 20 amino acids found in proteins are shown in the Figure at the end of this pr — Physical Chemistry — Kinetics Chemistry Question
Mechanism of Catalysis by Lactate Dehydrogenase
The structures of the 20 amino acids found in proteins are shown in the Figure at the end of this problem. [VISUAL]
The enzyme lactate dehydrogenase (LDH) catalyzes the reversible reduction of pyruvate anion to lactate anion, with NADH as the reducing agent. The reaction is formally the transfer of hydride ion (H ¯ ) from NADH to pyruvate:
[VISUAL]
The enzyme also catalyzes a reaction of sulfite (SO3 2–) and NAD+:
[VISUAL]
The structure of the substrates pyruvate and NADH bound in the active site of LDH is shown schematically in Scheme 1. Several key amino acid residues in the active site are indicated. The dotted lines between fragments of LDH indicate weak intermolecular interactions among groups in the active site.
Scheme 1
[VISUAL]
The pH dependence of the rate of the reactions catalyzed by LDH was determined with pyruvate and NADH as the substrates for the forward reaction, and with lactate and NAD+ as the substrates for the reverse reaction. The data indicate the participation in catalysis of a group with pKa = 7, which corresponds to His-195 of LDH.
The pH vs. reaction rate curves were different depending on whether the rate of the forward (pyruvate + NADH) or reverse (lactate + NAD+) reaction was measured, as shown in Figure below.
[VISUAL]
Which curve in the Figure above corresponds to the reaction with pyruvate and NADH? Which curve corresponds to the reaction with lactate and NAD+?
Model Answer
Curve A is the reaction with pyruvate, and curve B is the reaction with lactate. Since the reaction with pyruvate involves acid as a reagent, at lower pH values one might expect faster reaction rates. According to Scheme 1, His-195 acts as an acid, by donating a proton to pyruvate. Thus, for the reaction with pyruvate, His-195 must be protonated at the start of the reaction. His-195 will be protonated at low pH, and the rate of the reaction will be highest at low pH, as in curve A.
For the reverse reaction, His-195 acts as a base to remove a proton from lactate. In this case, His-195 must be deprotonated at the start of the reaction. The reaction rate is greatest at high pH, as in curve B.
As shown in Scheme 1, the side chains of Arg-109 and His-195 are very close to the carbonyl group of pyruvate.
What type of weak intermolecular interactions exists between Arg-109 and the carbonyl group of pyruvate, and between His-195 and the carbonyl group of pyruvate? What is the electronic basis of this interaction?
Model Answer
Hydrogen bonds. The carbonyl group is polarized, with a partial negative charge on the oxygen. The partial negative charge is attracted electrostatically to the positively charged His-195 and Arg-109.
The side chain of Ile-250 lies directly below the plane of the dihydronicotinamide ring of NADH (Scheme 1).
What type of intermolecular interaction would the side chain of Ile-250 make with NADH?
Model Answer
Ile-250 can be involved in dispersion interactions with the dihydronicotinamide ring. The instantaneous dipole in Ile-250 that arises due to spontaneous fluctuations of electron density induces a dipole in the dihydronicotinamide, and the two dipoles interact.
The function of Arg-109 in catalysis by LDH was investigated by site-directed mutagenesis. Arg-109 was changed to glutamine, and the catalytic activity of the mutant enzyme was studied. The results were:
- The rate of the (pyruvate + NADH) reaction catalyzed by the mutant enzyme was 1400-fold less than the reaction catalyzed by the wild-type enzyme.
- The ability of the mutant enzyme to bind pyruvate in the active site was also reduced, but by only about 15-fold compared to the wild-type enzyme.
- The rate of the reaction of sulfite with NAD+ was unaffected by the mutation.
Given the observations above, what is the function of Arg-109 in catalysis by LDH?
Model Answer
The Arg-109 end group increases the polarization of the carbonyl group of pyruvate, making it more susceptible to reaction with hydride. This accounts for the great decrease in reaction rate when Arg-109 is mutated to glutamine. This polarization is irrelevant for the sulfite reaction, so that reaction is unaffected by the mutation. Arg-109 is unlikely to be the acid that protonates pyruvate, since it is a very weak acid (pKa ~ 12.5). His-195 is more acidic (see above).
Arg-109 also seems to assist in binding pyruvate in the active site, as indicated in the figure and reflected in the 15-fold reduction in binding affinity for pyruvate.
The side chain of Asp-168 is thought to interact non-covalently with the side chain of His-195 (see Scheme 1). Two hypotheses were proposed for the function of Asp-168 in catalysis by LDH:
1) The interaction between Asp-168 and His-195 might serve to hold the His-195 in the correct position to interact with pyruvate.
2) The interaction between Asp-168 and His-195 might serve to polarize His-195, which would make His-195 a stronger base.
To test these possibilities Asp-168 was changed to Ala (Mutant 2), and to Asn (Mutant 1), and the catalytic properties of the mutant enzymes were compared to those of the wild-type enzyme.
The results are summarized in the following table:
| Constant | Wild-type (Asp-168) | Mutant 1 (Asn-168 ) | Ratio: Wild-type / Mutant 1 | Mutant 2 (Ala-168 ) | Ratio: Wild-type / Mutant 2 |
|---|---|---|---|---|---|
| Forward reaction: | | | | | |
| Km (pyruvate), mol dm–3 × 10–3 | 0.06 | 10 | 0.006 | 3.3 | 0.018 |
| kcat, s–1 | 250 | 20 | 12.5 | 5.5 | 45 |
| kcat/Km, mol–1 dm3 s–1 | 4.2·106 | 2·103 | 2080 | 1.7·103 | 2500 |
| Reverse reaction: | | | | | |
| Km (lactate), mol dm–3 × 10–3 | 40 | 120 | 0.33 | 80 | 0.5 |
| kcat, s–1 | 9 | 0.12 | 75 | 0.09 | 100 |
| kcat/Km, mol–1 dm3 s–1 | 2.2·102 | 1 | 225 | 1.13 | 200 |
Given the facts above, which of the proposed functions, (1) or (2), of Asp-168 is better supported by the data?
Model Answer
The conclusion from this set of experiments is that hydrogen bonding between Asp-168 and His-195 must polarize His-195 and make it a stronger base than it would be otherwise. This is reflected in the fact that the pKa of His-195 = 7 (see above), is somewhat greater than expected for a His residue (pKa for His is ~6.0).
Asn should be able to form a hydrogen bond to His-195 similar to that of Asp, but that would be a weaker bond since Asn side chain is neutral whereas it is negatively charged in Asp. On the other hand, Ala is unable to form such a hydrogen bond. If the only function of Asp-168 were to hold His-195 in the proper orientation for reaction, then one would predict that the Asn mutant would be significantly more active than the Ala mutant, which is not observed.