Opening Image: Solvent-accessible surface of human triose-phosphate isomerase. The two identical subunits are color-coded in blue and purple. The competitive inhibitor, phosphoglycolate, is displayed in spacefilling model; it is tightly bound at the active site of one subunit (chain B, in this case).

Outline

Introduction

Triose-phosphate isomerase (TIM) is a glycolytic enzyme that catalyzes the interconversion of two sugar triose phosphates, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate.

Glucose metabolism is very important. All TIM mutations reported to date retain some enzymatic activity. It's believed that mutations resulting in complete loss of enzymatic activity are lethal in the embryo. The most common mutation in TIM is the substitution of Glu-104 by Asp. Compared with wild type, the catalytic efficiency of the Glu104Asp mutant is unchanged, but its stability is considerably reduced, resulting in progressive, usually fatal neuromuscular dysfunction. It's hard to imagine how the loss of a side-chain methylene group (–CH₂CH₂COO^- to –CH₂COO^-) could cause a molecular disease.

What are the structural consequences resulting from the replacement of Glu-104 with Asp-104? To answer this question, we need to understand the molecular "glue" that holds the two subunits together.

Subunit Interface in the Dimer

The enzyme is a dimer of two identical subunits. In other words, it's a homodimer. The presence of a deep water-filled gap, or "cave", at the subunit interface creates a horseshoe-shaped contact surface between the two subunits. Despite the presence of this gap, the two monomers associate very strongly with each other and dissociation does not occur under physiological conditions. The basis for this strong association is the precise shape and electronic complementarity of the interface.

chain B. The atoms of chain A that make contact with chain B are colored purple.

In this brief lesson we explore the salt bridges at the subunit interface in the TIM dimer. For most salt bridges, the desolvation cost for bridge formation is paid off primarily by the electrostatic interaction of the salt-bridging residues with each other. Residues involved in surface salt bridges are expected to pay lower desolvation energy penalties as compared to those involved in buried salt bridges. Therefore, the surface salt bridges described next play a crucial role in the formation and stability of the TIM dimer.

Charged Residues at the Interface

Backbone trace of chain B. Note how the chain wanders into various "holes" and wraps around several "knobs" on the opposite subunit. spacefill.

As noted in the previous tutorial, the shape of the binding sites in small homodimeric proteins is usually quite flat. However, for TIM the polypeptide chains wrap around each other at the interface, suggesting that the entire dimer forms in one folding step, instead of from the association of two individually folded subunits.

acidic residues red. Identify the glutamate residues lining the inside wall of the cave. Are there any aspartates in the cave? []

basic residues blue. What do you think is a ball-park figure for the net charge of the dimer at physiological pH? []

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Careful inspection reveals about the same number of side-chain carboxyls (red) and arginine and lysine side-chains (blue). Thus the net charge is close to zero.

Since the pK of the imidazole group of histidine is around 6.5, histidine residues are not likely to fully ionized at physiological pH. Furthermore, histidine is relatively scare in proteins, compared to lysine and arginine. So histidines seldom make a large contribution to the positive charge of large proteins.

For human triose-phosphate isomerase, there are a total of 58 Asp and Glu residues, 56 Arg and Lys residues, and only 4 His residues. Assuming that the hisitidines are full charged, the net charge on the dimer would be +2 at pH 7. The N-terminal amino and C-terminal carboxyl groups of each chain are essentially fully charged at pH 7 and cancel each other.

Interchain Salt Bridges

Negatively charged (red) and positively charged (blue) sidechains of chain A which are located at the subunit interface.

Interchain salt bridges at the subunit interface.

The Lys18•Asp49 interchain salt bridge. The narrow channel in the center of the interface extends through the molecule and is aligned with the two-fold symmetry axis of the dimer. [Note: Protein symmetry is discussed in the next tutorial.]

Lys18•Asp49 interchain salt bridge up close. α carbons are shown in dark gray.

Two Glu77•Arg98 interchain salt bridges line the inside wall of the gap between the subunits. [The model has been "slabbed" along a plane so that we can view inside.]

When arginine is a partner in a salt bridge, the guanidino group can be positioned more precisely through additional noncovalent interactions. For example, in the Glu77A•Arg98B salt bridge, the guanidino group of Arg98B is also hydrogen bonded to the backbone carbonyl oxygen of Thr75A.

Close up of the Arg98•Glu77 interchain salt bridge. spacefill.

Salt Bridge Network

Up to now, we have focused on interchain salt bridges. However, one of the interchain ionic interactions discussed above is actually part of a network of both inter- and intra-chain ionic interactions. Although the details are fascinating, the important thing is to come away from this tutorial with an appreciation of the big picture.

The side-chain carboxyl of Glu-104B is clamped between two positively charged residues, Arg-98B and Lys-112B, of the same chain. There is an identical set of interactions in chain A. spacefill.

There is also a hydrogen bond between the side-chain amide group of Asn-65B and Glu-104B. spacefill.

Five side chains are interlocked by noncovalent interactions. Although only two side chains, Glu-77A and Arg-98B (and the corresponding Glu-77B•Arg-98A pair) form interchain salt bridges, the intrachain interactions play an important role in maintaining the interchain salt linkages. spacefill.

In molecular terms, the interactions shown here are cooperative, that is, the overall interaction energy is greater than the sum of the individual interactions. That, of course, is true for most of the interactions which stabilize tertiary and quaternary structure.

Reduced Stability of the Mutant

The crystallographic structure of the human enzyme was solved thirty years after the discovery of the Glu104Asp mutation. JSmol is able to simulate the mutation. The structure shows us how the replacement of Glu-104 by Asp is sufficient to disrupt the key interactions discussed this far.

Glu-104 with Asp-104. [JSmol may take a moment to process. For the clearest view, don't move the camera until the process is complete.]

spacefill.

With respect to to the guanidino group of Arg-98, Asp-104 is positioned too far away for the two to form an intrachain salt bridge without disrupting the interchain salt bridge between Arg-98A and Glu-77B (or Arg-98B and Glu-77A). Furthermore, Asp-104 no longer lies within hydrogen-bonding distance of Asn-65 and Lys-112.

Triose-phosphate isomerase is a highly tuned enzyme, with a catalytic efficiency approaching the theoretical limit for enzyme-catalyzed reactions. Thus, it's not surprising that the substitution of Glu-104 by Asp affects the stability of TIM. In fact, decreased protein stability is often a disease-causing factor.

The TIM Barrel

Before you leave this tutorial, take a moment to explore the secondary structure of a TIM monomer. The small molecule shown in CPK colors is the competitive inhibitor, 2-phosphoglycolic acid. It is tightly bound at the active site, which is located at one end of a β-barrel. The β-barrel, first observed in triose-phosphate isomerase, occurs ubiquitously in nature. It is nearly always an enzyme.

cartoon representation. The β-sheet is an eight-stranded, all-parallel sheet. This α/β fold, often called a TIM barrel, is the most common fold among protein catalysts, appearing in approximately 10% of all known enzyme structures.