Channeling enzymes

In our cells, as in the laboratory, the conversion of reactant molecules to a particular product may proceed in multiple steps, so that there are intermediate steps and intermediate substrates. In the laboratory, we are usually able to control the sequence of events and we are able to isolate the various intermediates and pass them on to the next step. We have no such control over the chemical reactions in our cells. In our cells, the movement of molecules may be by simple diffusion, in other words, in a random direction, with speeds that depend primarily on the properties of the surrounding medium. However, to increase the probability of chemical reactions taking place at high speed and in the right direction, Nature has evolved ingenious ways of directing the movement of substrate molecules in cells.

One way is to assemble the enzymes, which catalyze consecutive reactions, in close proximity to each other. In that situation, the intermediates have a higher probability of finding the right active sites. Nature does this in several ways. One is by forming an enzyme cluster, or a multi-subunit enzyme with each subunit performing a separate step in the chemical reaction. The subunits would be in tight contact to prevent them from drifting apart. Nature has also linked the various subunits covalently so that they are tethered to each other and cannot drift apart.

But Nature has found ways to be even more efficient. It actually directs the movement of substrate intermediates from one active site to the next. Such enzymes are called “channeling enzymes.” There are several types.

The very first channeling enzyme whose three-dimensional structure was determined and whose mode of action was explained in structural terms is tryptophan synthase (Hyde et al. 1988). From its name, the function of tryptophan synthase is to catalyze the synthesis of tryptophan. The enzyme does it in two steps. First, using indole glycerol phosphate as substrate, it strips off the indole moiety. The indole is then reacted with a serine to form tryptophan. The reaction is summarized below.

First reaction:

Indole 3-glycerol-P < = > Indole + D-glyceraldehyde-3-P

Second reaction:

Indole + L-serine L-tryptophan + H20

Overall reaction:

Indole 3-glycerol-P + L-serine L-tryptophan + D-glyceraldehyde-3-P + H20

Clearly, tryptophan synthase has to have two active sites. So, how does Nature “channel” the indole intermediate from the first site to the second? For one thing, indole is quite hydrophobic and if released into the (aqueous) cytoplasm, it will immediately head for the (lipid) cell membrane and, consequently, will no longer be available for the next step of the reaction. So, Nature has fashioned a hydrophobic “tunnel,” 25 Angstroms long and closed to the outside, which links the two active sites. Very clever!

Tryptophan synthase is not the only “tunneling enzyme.” Another example is carbamoyl phosphate synthetase, which catalyzes four reactions. In this enzyme complex, the three reactive intermediates are shuttled between the first and last active sites by a 96-Angstrom tunnel (Thoden et al. 1997).

But substrate transfer need not involve a tunnel. An open channel might suffice.

An example is provided by the bifunctional enzyme, dihydrofolate reductase-thymidylate synthase from Leishmania major. It catalyzes a two-step reaction, involving a negatively charged intermediate. The intermediate is channeled between the two active sites by a 40-Angstrom positively charged electrostatic “highway” on the surface of the enzyme complex (Knighton et al. 1994).

There are other examples of enzymes that guide the intermediate(s) from one active site to another using channels/tunnels.

But Nature has devised yet another way to make an efficient transfer of intermediates. For example, pyruvate phosphate dikinase catalyzes a two-step reaction and its two active sites are normally 45 Angstroms apart. But when an intermediate is present in the first site, a “swiveling motion” involving flexible parts of the enzyme brings the two active sites closer to each other, allowing the easy transfer of the intermediate between the two sites (Herzberg et al. 1996).

One wonders what other clever means Nature uses to make biological reactions more efficient and more productive.

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References:

Herzberg O, Chen CC, Kapadia G, McGuire M, Carroll LJ, Noh SJ, Dunaway-Mariano D. Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites. Proc Natl Acad Sci USA 1996; 93(7):2652-2657.

Hyde CC, Ahmed SA, Padlan EA, Miles EW, Davies DR. Three-dimensional structure of the tryptophan synthase _2_2 multienzyme complex from Salmonella typhimurium. J Biol Chem 1988; 263(33):17857-17871.

Knighton DR, Kan C-C, Howland E, Janson CA, Hostomska Z, Welsh KM, Matthews DA. Structure of and kinetic channelling in bifunctional dihydrofolate reductase-thymidylate synthase. Nature Structural Biology 1994; 1:186-194.

Thoden JB, Holden HM, Wesenberg G, Raushel FM, Rayment I. Structure of carbamoyl phosphate synthetase: a journey of 96 A from substrate to product. Biochemistry. 1997; 36(21):6305-6316.

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Eduardo A. Padlan is an adjunct professor at the Marine Science Institute, UP Diliman, and is a corresponding member of the NAST. He can be reached at [email protected].