Dihydroxyacetone phosphate, binding

Figure 8.22. Bromoacetol Phosphate, an Affinity Label for Triose Phosphate Isomerase (TIM). Bromoacetol phosphate, an analog of dihydroxyacetone phosphate, binds at the active site of the enzyme and covalently modifies a glutamic acid residue required for enzyme activity.

Enzymes are very sophisticated systems that apply sound chemical principles. The side-chains of various amino acids are used to supply the necessary bases and acids to help catalyse the reaction (see Section 13.4). Thus, the enzyme aldolase binds the dihydroxyacetone phosphate substrate by reacting the ketone group with an amine, part of a lysine amino acid residue. This forms an imine that becomes protonated under normal physiological conditions. 

The calculated free energy profile of the reaction catalyzed by triosephosphate isomerase. (Enz = enzyme DHAP = dihydroxyacetone phosphate GAP = glyceraldehyde-3-phosphate.) The free energy changes associated with binding of DHAP and GAP to the enzyme are calculated on the assumption that DHAP and GAP are present at concentrations of 40 /um. 

The majority of useful lyase families utilize anionically functionalized substrates such as pyruvate or dihydroxyacetone phosphate which remain unaltered during catalysis. The charged group thereby introduced into the products (phosphate, carboxylate) not only constitutes a handle for binding of the substrates by the enzymes but also can facilitate the preparative isolation from... 

Free fatty acids may be absorbed directly by tissues, or bound to albmnin for transport human serum albmnin possesses multiple fatty acid binding sites of various affinities. Glycerol is returned via the blood to the liver (and kidneys), where it is converted to the glycolytic intermediate dihydroxyacetone phosphate (glycerol is an important source of glucose in gluco-... 

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They are thus more specific for the enzyme active site than are group-specific reagents. Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analog for chymotrypsin (Figure 8.21). TPCK binds at the active site and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme. The compound 3-bromoacetol is an affinity label for the enzyme triose phosphate isomerase (TIM). It mimics the normal substrate, dihydroxyacetone phosphate, by binding at the active site then it covalently modifies the enzyme such that the enzyme is irreversibly inhibited (Figure 8.22). 

An enzyme that has been the subject of intensive experimental and theoretical studies is triosephosphate isomerase (TIM), which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP), an essential step in the glycolytic pathway (Fersht 1985). The mechanism of the enzyme has been examined by QM/MM calculations which we do not describe here because it falls outside the topic ofthis review (Bash et al. 1991). However, an additional aspect of the overall mechanism is the conformational change of an 11-residue loop region (residues 166-176) which moves more than 7 A and closes over the active site when substrate binds (Joseph et al. 1990 Lolis et al. 1990). Mutagenesis experiments have... 

Triose phosphate isomerase enzyme catalyses interconversion of the 3-carbon triose phosphate dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (D-GAP). The reaction is just the transfer of the pro-R hydrogen from carbon 1 of DHAP stereospecifically to carbon 2 to form the D-isomer of GAP (Figure 2). Although the equilibrium constant on the enzyme is not known, Keq for the overall reaction is 300 to 1 in favour of DHAP. The large magnitude of this number arises from the combination of an apparent Keq of 22 vith a hydration equilibrium of 29 for the hydrated and unhydrated forms of D-GAP (Trentham ei al., 1969) only the unhydrated forms of the triose phosphates are substrates for or even bind to the isomerase (Vebb eial., 1977). 

Fig. 4. Ether phospholipid synthesis from dihydroxyacetone-phosphate. (A) Dihydroxyacetone-P acyl transferase (DHAPAT). The first step of ether phospholipid synthesis is catalyzed by peroxisomal DHAPAT. This enzyme is a required component of complex ether lipid biosynthesis and its role cannot be assumed by a cytosolic enzyme that also forms acyldihydroxyacetone-P. (B) Ether bond formation by alkyl-DHAP synthase. The reaction that forms the 0-alkyl bond is catalyzed by alkyl-DHAP synthase and is thought to proceed via a ping-pong mechanism. Upon binding of acyl-DHAP to the enzyme alkyl-DHAP synthase, the pro-f hydrogen at carbon atom 1 is exchanged by enolization of the ketone, followed by release of the acyl moiety to form an activated enzyme-DHAP complex. The carbon atom at the 1-position of DHAP in the enzyme complex is thought to carry a positive charge that may be stabilized by an essential sulfhydryl group of the enzyme thus, the incoming alkox-ide ion reacts with carbon atom 1 to form the ether bond of alkyl-DHAP. It has been proposed that a nucleophilic cofactor at the active site covalently binds the DHAP portion of the substrate.

Gholson et al. (1976) have studied quinolinic acid (QA) biosynthesis, in a cell-free system prepared from E. coli mutants. In this system QA is synthesized by the condensation of aspartate and a [3- C]dihydroxyacetone phosphate which is incorporated into the C-4 of QA. An FAD-requiring reaction is catalyzed by two partially purified proteins which they call quinolinate synthetase. Quinolinate synthetase is composed of protein A (MW about 35,(X)0) and protein B(MW about 85,000). Preincubation of A and B proteins leads to inactivation of at least the A protein and DHAP prevents this inactivation reaction. Neither the A nor the B protein binds aspartate- C but in the presence of both proteins aspartate is bound to an entity with an apparent MW greater than the B protein. 

The first of these two enzymes, which was found in the c3 oplasm accepts the hydrogen from glycerol-2- H which is transformed into P-dihydroxyacetone, and binds it to NAD. This is a reaction of secondary importance as the equilibrium is strongly shifted toward glycerol phosphate (Boxer and Shonk, 1960). 

Another class of potential inhibitors is the haloacetol phosphates of bromine, chlorine, and iodine. Hartman showed that there was irreversible binding of these compounds to the substrate active site of triose-phos-phate isomerase because of structural similarities to dihydroxyacetone phosphate.Subsequent work with isolated wheat chloroplasts has indicated that iodoacetol phosphates enter the chloroplast by the phosphate translocator and primarily inhibit glyceraldehyde-3-phosphate dihydro-... 

Garrabou, X., Joglar, J., Parella, T., Bujons, J., and Clapes, R, Redesign of the phosphate binding site of L-rhamnulose-1-phosphate aldolase towards a dihydroxyacetone dependent aldolase. Adv. Synth. Catal. 2011,353 (1), 89-99. 

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